Engineered Nucleic-Acid Targeting Nucleic Acids

ABSTRACT

The present disclosure provides engineered cross-type-nucleic-acid targeting nucleic acids and compositions thereof. Nucleic acid sequences encoding the engineered cross-type-nucleic-acid targeting nucleic acids, as well as expression cassettes, vectors and cells comprising such nucleic acid sequences, are described. Also, methods are disclosed for making and using the engineered cross-type-nucleic-acid targeting nucleic acids and compositions thereof.

The present application is a Continuation of U.S. patent applicationSer. No. 16/224,747, filed 18 Dec. 2018, now allowed, which is aContinuation of U.S. patent application Ser. No. 16/166,097, filed 20Oct. 2018, now U.S. Pat. No. 10,196,619, which is a Continuation of U.S.patent application Ser. No. 16/036,599, filed 16 Jul. 2018, now U.S.Pat. No. 10,138,472, which is a Continuation of U.S. patent applicationSer. No. 15/919,202, filed 12 Mar. 2018, now U.S. Pat. No. 10,023,853,which is a Continuation of U.S. patent application Ser. No. 15/787,705,filed 18 Oct. 2017, now U.S. Pat. No. 9,957,490, which is a Continuationof U.S. patent application Ser. No. 15/675,677, filed 11 Aug. 2017, nowU.S. Pat. No. 9,816,081, which is a Continuation of U.S. patentapplication Ser. No. 15/460,642, filed on 16 Mar. 2017, now U.S. Pat.No. 9,745,562, which is a Continuation of U.S. patent application Ser.No. 15/331,676, filed on 21 Oct. 2016, now U.S. Pat. No. 9,677,090,which claims the benefit of U.S. Provisional Application Serial No.62/245,918, filed 23 Oct. 2015, now expired.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/245,918, filed 23 Oct. 2015, now pending, whichapplication is herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

SEQUENCE LISTING

The present application contains a Sequence Listing that has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on 15 Oct. 2016 isnamed CBI019-10 ST25.txt and is 9 KB in size.

TECHNICAL FIELD

The present disclosure relates generally to engineered nucleic-acidtargeting nucleic acids and nucleoprotein complexes comprising one ormore Cas proteins and one or more engineered nucleic-acid targetingnucleic acids. The disclosure also relates to compositions and methodsfor making and using the engineered nucleic-acid targeting nucleic acidsand nucleoprotein complexes of the present invention.

BACKGROUND

Clustered regularly interspaced short palindromic repeats (CRISPR) andCRISPR-associated proteins (Cas) constitute the CRISPR-Cas system. TheCRISPR-Cas system provides adaptive immunity against foreign DNA inbacteria (see, e.g., Barrangou, R., et al., Science 315:1709-1712(2007); Makarova, K. S., et al., Nature Reviews Microbiology 9:467-477(2011); Garneau, J. E., et al., Nature 468:67-71 (2010); Sapranauskas,R., et al., Nucleic Acids Research 39:9275-9282 (2011)).

CRISPR-Cas systems have recently been reclassified into two classes,comprising five types and sixteen subtypes (see Makarova, K., et al.,Nature Reviews Microbiology 13:1-15 (2015)). This classification isbased upon identifying all cas genes in a CRISPR-Cas locus anddetermining the signature genes in each CRISPR-Cas locus, ultimatelydetermining that the CRISPR-Cas systems can be placed in either Class 1or Class 2 based upon the genes encoding the effector module, i.e., theproteins involved in the interference stage. Recently a sixth CRISPR-Cassystem (Type VI) has been identified (see Abudayyeh O., et al., Science353(6299):aaf5573 (2016)). Certain bacteria possess more than one typeof CRISPR-Cas system.

Class 1 systems have a multi-subunit crRNA-effector complex, whereasClass 2 systems have a single protein, such as Cas9, Cpf1, C2c1, C2c2,C2c3, or a crRNA-effector complex. Class 1 systems comprise Type I, TypeIII, and Type IV systems. Class 2 systems comprise Type II and Type Vsystems.

Type II systems have cas1, cas2, and cas9 genes. The cas9 gene encodes amulti-domain protein that combines the functions of the crRNA-effectorcomplex with DNA target sequence cleavage. Type II systems also encode atracrRNA. Type II systems are further divided into three subtypes,subtypes II-A, II-B, and II-C. Subtype II-A contains an additional gene,csn2. Examples of organisms with a subtype II-A systems include, but arenot limited to, Streptococcus pyogenes, Streptococcus thermophilus, andStaphylococcus aureus. Subtype II-B lacks csn2, but has cas4. An exampleof an organism with a subtype II-B system is Legionella pneumophila.Subtype II-C is the most common Type II system found in bacteria and hasonly three proteins, Cas1, Cas2, and Cas9. An example of an organismwith a subtype II-C system is Neisseria lactamica.

Type V systems have a cpf1 gene and cast and cas2 genes (see Zetsche,B., et al., Cell 163:1-13 (2015)). The cpf1 gene encodes a protein,Cpf1, that has a RuvC-like nuclease domain that is homologous to therespective domain of Cas9, but lacks the HNH nuclease domain that ispresent in Cas9 proteins. Type V systems have been identified in severalbacteria including, but not limited to, Parcubacteria bacterium,Lachnospiraceae bacterium, Butyrivibrio proteoclasticus,Peregrinibacteria bacterium, Acidaminococcus spp., Porphyromonasmacacae, Porphyromonas crevioricanis, Prevotella disiens, Moraxellabovoculi, Smithella spp., Leptospira inadai, Franciscella tularensis,Franciscella novicida, Candidatus methanoplasma termitum, andEubacterium eligens. Recently it has been demonstrated that Cpf1 alsohas RNase activity, and it is responsible for pre-crRNA processing (seeFonfara, I., et al., Nature 532(7600):517-521 (2016)).

In Class 2 systems, the crRNA is associated with a single protein andachieves interference by combining nuclease activity with RNA-bindingdomains and base-pair formation between the crRNA and a nucleic acidtarget sequence.

In Type II systems, nucleic acid target sequence binding involves Cas9and the crRNA, as does the nucleic acid target sequence cleavage. InType II systems, the RuvC-like nuclease (RNase H fold) domain and theHNH (McrA-like) nuclease domain of Cas9 each cleave one of the strandsof the double-stranded nucleic acid target sequence. The Cas9 cleavageactivity of Type II systems also requires hybridization of crRNA totracrRNA to form a duplex that facilitates the crRNA and nucleic acidtarget sequence binding by the Cas9.

In Type V systems, nucleic acid target sequence binding involves Cpf1and the crRNA, as does the nucleic acid target sequence cleavage. InType V systems, the RuvC-like nuclease domain of Cpf1 cleaves one strandof the double-stranded nucleic acid target sequence, and a putativenuclease domain cleaves the other strand of the double-stranded nucleicacid target sequence in a staggered configuration, producing 5′overhangs, which is in contrast to the blunt ends generated by Cas9cleavage. These 5′ overhangs may facilitate insertion of DNA.

The Cpf1 cleavage activity of Type V systems also does not requirehybridization of crRNA to tracrRNA to form a duplex, rather the crRNA ofType V systems uses a single crRNA that has a stem-loop structureforming an internal duplex. Cpf1 binds the crRNA in a sequence andstructure specific manner that recognizes the stem loop and sequencesadjacent to the stem loop, most notably the nucleotide 5′ of the spacersequences that hybridizes to the nucleic acid target sequence. Thisstem-loop structure is typically in the range of 15 to 19 nucleotides inlength. Substitutions that disrupt this stem-loop duplex abolishcleavage activity, whereas other substitutions that do not disrupt thestem-loop duplex do not abolish cleavage activity. In Type V systems,the crRNA forms a stem-loop structure at the 5′ end, and the sequence atthe 3′ end is complementary to a sequence in a nucleic acid targetsequence.

Other proteins associated with Type V crRNA and nucleic acid targetsequence binding and cleavage include Class 2 candidate 1 (C2c1) andClass 2 candidate 3 (C2c3). C2c1 and C2c3 proteins are similar in lengthto Cas9 and Cpf1 proteins, ranging from approximately 1,100 amino acidsto approximately 1,500 amino acids. C2c1 and C2c3 proteins also containRuvC-like nuclease domains and have an architecture similar to Cpf1.C2c1 proteins are similar to Cas9 proteins in requiring a crRNA and atracrRNA for nucleic acid target sequence binding and cleavage but havean optimal cleavage temperature of 50° C. C2c1 proteins target anAT-rich protospacer adjacent motif (PAM), which similar to the PAM ofCpf1, is 5′ of the nucleic acid target sequence (see, e.g., Shmakov, S.,et al., Molecular Cell 60(3):385-397 (2015)).

Class 2 candidate 2 (C2c2) does not share sequence similarity to otherCRISPR effector proteins and was recently identified as a Type VI system(see Abudayyeh, O., et al., Science 353(6299):aaf5573 (2016)). C2c2proteins have two HEPN domains and demonstrate single-strandedRNA-cleavage activity. C2c2 proteins are similar to Cpf1 proteins inrequiring a crRNA for nucleic acid target sequence binding and cleavage,although not requiring tracrRNA. Also similar to Cpf1, the crRNA forC2c2 proteins forms a stable hairpin, or stem-loop structure, that aidsin association with the C2c2 protein. Type VI is a single polypeptideRNA endonuclease that utilizes a single crRNA to direct site specificcleavage. Additionally, after hybridizing to the target RNAcomplementary to the spacer, C2c2 becomes a promiscuous RNA endonucleaseexhibiting non-specific endonuclease activity toward any single-strandedRNA in a sequence independent manner (see East-Seletsky, A., et al.,Nature 538(7624):270-273 (2016)).

Regarding Class 2 Type II CRISPR-Cas systems, a large number of Cas9orthologs are known in the art as well as their associatedpolynucleotide components (tracrRNA and crRNA) (see, e.g., Fonfara, I.,et al., Nucleic Acids Research 42(4):2577-2590 (2014), including allSupplemental Data; Chylinski K., et al., Nucleic Acids Research42(10):6091-6105 (2014), including all Supplemental Data). In addition,Cas9-like synthetic proteins are known in the art (see U.S. PublishedPatent Application No. 2014-0315985, published 23 Oct. 2014).

Cas9 is an exemplary Type II CRISPR Cas protein. Cas9 is an endonucleasethat can be programmed by the tracrRNA/crRNA to cleave,site-specifically, a DNA target sequence using two distinct endonucleasedomains (HNH and RuvC/RNase H-like domains) (see U.S. Published PatentApplication No. 2014-0068797, published 6 Mar. 2014; see also Jinek M.,et al., Science 337:816-821 (2012)).

Typically, each wild-type CRISPR-Cas9 system includes a crRNA and atracrRNA. The crRNA has a region of complementarity to a potential DNAtarget sequence and a second region that forms base-pair hydrogen bondswith the tracrRNA to form a secondary structure, typically to form atleast a stem structure. The region of complementarity to the DNA targetsequence is the spacer. The tracrRNA and a crRNA interact through anumber of base-pair hydrogen bonds to form secondary RNA structures.Complex formation between tracrRNA/crRNA and Cas9 protein results inconformational change of the Cas9 protein that facilitates binding toDNA, endonuclease activities of the Cas9 protein, and crRNA-guidedsite-specific DNA cleavage by the endonuclease Cas9. For a Cas9protein/tracrRNA/crRNA complex to cleave a double-stranded DNA targetsequence, the DNA target sequence is adjacent to a cognate PAM. Byengineering a crRNA to have an appropriate spacer sequence, the complexcan be targeted to cleave at a locus of interest, e.g., a locus at whichsequence modification is desired.

Ran, F. A., et al., Nature 520(7546):186-191 (2015), including allextended data, present the crRNA/tracrRNA sequences and secondarystructures of eight Type II CRISPR-Cas systems (see Extended Data Figure1 of Ran, F.A ., et al.). Predicted tracrRNA structures were based onthe Constraint Generation RNA folding model (Zuker, M., Nucleic AcidsResearch 31:3406-3415 (2003)). Furthermore, Fonfara, et al., NucleicAcids Research 42(4):2577-2590 (2014), including all Supplemental Data(in particular Supplemental Figure S11) present the crRNA/tracrRNAsequences and secondary structures of eight Type II CRISPR-Cas systems.RNA duplex secondary structures were predicted using RNAcofold of theVienna RNA package (Bernhart, S. H., et al., Algorithms for MolecularBiology 1(1):3 (2006); Hofacker, I. L., et al., Journal of MolecularBiology 319:1059-1066 (2002)) and RNAhybrid(bibiserv.techfak.uni-bielefeld.de/rnahybrid/). The structurepredictions were visualized using VARNA (Darty, K., et al.,Bioinformatics 25:1974-1975 (2009)). Fonfara, et al., show that thecrRNA/tracrRNA complex for Campylobacter jejuni does not have the bulgeregion; however, it retains a stem structure located 3′ of the spacerthat is followed in the 3′ direction with another stem structure.

Naturally occurring Type V CRISPR-Cas systems, unlike Type II CRISPR Cassystems, do not require a tracrRNA for crRNA maturation and cleavage ofa nucleic acid target sequence. In a typical structure of a Type VCRISPR crRNA, the DNA target binding sequence is downstream of aspecific secondary structure (i.e., a stem-loop structure) thatinteracts with the Cpf1 protein. The bases 5′ of the stem loop adopt apseudo-knot structure further stabilizing the stem-loop structure withnon-canonical Watson-Crick base pairing, triplex interaction, andreverse Hoogsteen base pairing (see Yamano, T., et al., Cell165(4):949-962 (2016)).

The spacer of Class 2 CRISPR-Cas systems can hybridize to a nucleic acidtarget sequence that is located 5′ or 3′ of a PAM, depending upon theCas protein to be used. A PAM can vary depending upon the Caspolypeptide to be used. For example, if Cas9 from S. pyogenes is used,the PAM can be a sequence in the nucleic acid target sequence thatcomprises the sequence 5′-NRR-3′, wherein R can be either A or G,wherein N is any nucleotide, and N is immediately 3′ of the nucleic acidtarget sequence targeted by the nucleic acid target binding sequence. ACas protein may be modified such that a PAM may be different comparedwith a PAM for an unmodified Cas protein. For example, if Cas9 from S.pyogenes is used, the Cas9 protein may be modified such that the PAM nolonger comprises the sequence 5′-NRR-3′, but instead comprises thesequence 5′-NNR-3′, wherein R can be either A or G, wherein N is anynucleotide, and N is immediately 3′ of the nucleic acid target sequencetargeted by the nucleic acid target sequence.

Other Cas proteins recognize other PAMs, and one of skill in the art isable to determine the PAM for any particular Cas protein. For example,Cpf1 has a thymine-rich PAM site that targets, for example, a TTTNsequence (see Fagerlund, R., et al., Genome Biology 16:251 (2015)).

The RNA-guided Cas9 endonuclease has been widely used for programmablegenome editing in a variety of organisms and model systems (see, e.g.,Jinek M., et al., Science 337:816-821 (2012); Jinek M., et al., eLife2:e00471. doi: 10.7554/eLife.00471 (2013); U.S. Published PatentApplication No. 2014-0068797, published 6 Mar. 2014).

Genome engineering includes altering the genome by deleting, inserting,mutating, or substituting specific nucleic acid sequences. Thealteration can be gene- or location-specific. Genome engineering can usesite-directed nucleases, such as Cas proteins and their cognatepolynucleotides, to cut DNA, thereby generating a site for alteration.In certain cases, the cleavage can introduce a double-strand break (DSB)in the DNA target sequence. DSBs can be repaired, e.g., bynon-homologous end joining (NHEJ), microhomology-mediated end joining(MMEJ), or homology-directed repair (HDR). HDR relies on the presence ofa template for repair. In some examples of genome engineering, a donorpolynucleotide or portion thereof can be inserted into the break.

SUMMARY OF THE INVENTION

The present invention relates generally to engineeredcross-type-nucleic-acid targeting nucleic acids.

In one aspect the present invention relates to an engineered CRISPRClass 2 cross-type-nucleic-acid targeting nucleic acid (“CRISPR Class 2cross-type-NATNA”), comprising: a Cpf1-associated nucleic-acid targetingnucleic acid, having a 5′ end and a 3′ end, comprising a spacer element(“Cpf1-NATNA”); a first Cas9-associated nucleic-acid targeting nucleicacid, having a 5′ end and a 3′ end, comprising a spacer element (“firstCas9-NATNA”); and a second Cas9-associated nucleic-acid targetingnucleic acid, having a 5′ end and a 3′ end, comprising a tracr element(“second Cas9-NATNA”); wherein the first Cas9-NATNA or the secondCas9-NATNA is connected with the Cpf1-NATNA. In one embodiment, thefirst Cas9-NATNA or the second Cas9-NATNA is non-covalently connectedwith the Cpf1-NATNA, for example, through hydrogen base-pair bonding atthe 5′ end or 3′ end of the first Cas9-NATNA or through hydrogenbase-pair bonding at the 5′ end or 3′ end of the second Cas9-NATNA. Inan alternative embodiment, the first Cas9-NATNA or the second Cas9-NATNAis covalently connected with the Cpf1-NATNA, for example, the Cpf1-NATNAis covalently connected with the 5′ end or the 3′ end of the firstCas9-NATNA or to the 5′ end or the 3′ end of the second Cas9-NATNA.

In some embodiments, the Cpf1-NATNA of the CRISPR Class 2cross-type-NATNA is capable forming a first complex with a Cpf1 protein(“Cas9-Cpf1-NATNA/Cpf1 protein complex”), and the first Cas9-NATNA ofthe CRISPR Class 2 cross-type-NATNA and the second Cas9-NATNA of theCRISPR Class 2 cross-type-NATNA are capable of forming a second complexwith a Cas9 protein (“Cas9-Cpf1-NATNA/Cas9 protein complex”). If theCpf1-NATNA forms the first complex with the Cpf1 protein(“Cpf1-NATNA/Cpf1 protein complex”), the first complex is capable ofbinding a first double-stranded nucleic acid target sequencecomplementary to the Cpf1 spacer element. Furthermore, if the firstCas9-NATNA and the second Cas9-NATNA form the second complex with theCas9 protein (“Cas9-NATNA/Cas9 protein complex”), the second complex iscapable of binding a second double-stranded nucleic acid target sequencecomplementary to the first Cas9-NATNA spacer element. In otherembodiments, if the first complex is formed and the second complex isformed, resulting in a Cas9-Cpf1-NATNA/Cas9&Cpf1 protein complex, theCas9-Cpf1-NATNA/Cas9&Cpf1 protein complex is capable of binding a firstdouble-stranded nucleic acid target sequence complementary to the Cpf1spacer element and a second double-stranded nucleic acid target sequencecomplementary to the first Cas9-NATNA spacer element.

In further embodiments, one or more of the Cpf1-NATNA, the firstCas9-NATNA, and the second Cas9-NATNA can further comprise a linkerelement nucleotide sequence covalently connected with the 5′ end or the3′ end of the one or more of the Cpf1-NATNA, the first Cas9-NATNA, andthe second Cas9-NATNA. Furthermore, a first linker element nucleotidesequence and a second linker element nucleotide sequence can becovalently connected with both the 5′ and 3′ ends of one or more of theCpf1-NATNA, the first Cas9-NATNA, and the second Cas9-NATNA.

Embodiments of the present invention also include the engineered CRISPRClass 2 cross-type-NATNA, wherein the 3′ end of the first Cas9-NATNA iscovalently connected through a loop element with the 5′ end of thesecond Cas9-NATNA. The covalent connection results in asingle-Cas9-associated nucleic-acid targeting nucleic acid(“single-Cas9-NATNA”), having a 5′ end and a 3′ end. Thesingle-Cas9-NATNA comprises the first Cas9-NATNA and the secondCas9-NATNA. In some embodiments, the single-Cas9-NATNA is non-covalentlyconnected with the Cpf1-NATNA through hydrogen base-pair bonding at the5′ end or the 3′ end. Furthermore, a first Cpf1-NATNA and a secondCpf1-NATNA can be non-covalently connected with both the 5′ and 3′ endsof the single-Cas9-NATNA. In other embodiments, the single-Cas9-NATNA iscovalently connected with the Cpf1-NATNA. Additionally, a firstCpf1-NATNA and a second Cpf1-NATNA can be covalently connected with boththe 5′ and 3′ ends of the single-Cas9-NATNA.

In further embodiments, one or both of the Cpf1-NATNA and thesingle-Cas9-NATNA can further comprise a linker element nucleotidesequence covalently connected with the 5′ end or the 3′ end.Furthermore, a first linker element nucleotide sequence and a secondlinker element nucleotide sequence can be covalently connected with boththe 5′ and 3′ ends of one or both of the Cpf1-NATNA and thesingle-Cas9-NATNA.

In some embodiment, one or more of the Cpf1-NATNA, the first Cas9-NATNA,and the second Cas9-NATNA (including the single-Cas9-NATNA comprisingthe first Cas9-NATNA and the second Cas9-NATNA) of the engineered CRISPRClass 2 cross-type-NATNA can comprise RNA, DNA, or combinations of RNAand DNA.

In a further aspect, the present invention includes a nucleicacid/protein composition comprising an engineered CRISPR Class 2cross-type-NATNA, a Cas9 protein, and a Cpf1 protein. In someembodiments, the engineered CRISPR Class 2 cross-type-NATNA is in acomplex with the Cas9 protein and the Cpf1 protein. In additionalembodiments, one or both of the Cpf1 protein and the Cas9 protein can beenzymatically inactive. When the Cpf1 protein is enzymatically inactive(dCpf1 protein) or the Cas9 protein is enzymatically inactive (dCas9protein), the nucleic acid/protein composition can further comprises adonor polynucleotide non-covalently connected with the dCpf1 protein orthe dCas9 protein.

In another aspect, the present invention relates to an expressionvector, comprising one or more nucleic acid sequences encoding anengineered CRISPR Class 2 cross-type-NATNA.

In yet another aspect, the present invention includes a recombinantcell, comprising one or more nucleic acid sequences encoding anengineered CRISPR Class 2 cross-type-NATNA.

An additional aspect of the present invention includes a kit comprisingan engineered CRISPR Class 2 cross-type-NATNA and a buffer, or one ormore nucleic acid sequences encoding an engineered CRISPR Class 2cross-type-NATNA and a buffer. Kits can further comprise a Cas9 protein,a Cpf1 protein, or both a Cas9 protein and a Cpf1 protein. Furthermore,kits can comprise one or more nucleic acid sequences encoding a Cas9protein, a Cpf1 protein, or both a Cas9 protein and a Cpf1 protein.

Further aspects of the present invention include methods of using anengineered CRISPR Class 2 cross-type-NATNA, as described herein. Onemethod is a method of binding DNA. The method comprises contacting afirst DNA target sequence in the DNA and a second DNA target sequence inthe DNA with a nucleic acid/protein composition comprising an engineeredCRISPR Class 2 cross-type-NATNA, a Cas9 protein, and a Cpf1 protein;thereby facilitating binding of the nucleic acid/protein composition tothe first DNA target sequence in the DNA and the second DNA targetsequence in the DNA. The Cpf1-NATNA spacer element of the CRISPR Class 2cross-type-NATNA is complementary to the first DNA target sequence, andthe Cas9-NATNA spacer of the CRISPR Class 2 cross-type-NATNA iscomplementary to the second DNA target sequence.

Another method of the present invention is a method of cutting DNA. Themethod comprises contacting a first DNA target sequence in the DNA and asecond DNA target sequence in the DNA with a nucleic acid/proteincomposition comprising an engineered CRISPR Class 2 cross-type-NATNA, aCas9 protein, and a Cpf1 protein; thereby facilitating binding of thenucleic acid/protein composition to the first DNA target sequence andthe second DNA target sequence resulting in cutting of the first DNAtarget sequence and the second DNA target sequence. The Cpf1-NATNAspacer element of the CRISPR Class 2 cross-type-NATNA is complementaryto the first DNA target sequence, and the Cas9-NATNA spacer of theCRISPR Class 2 cross-type-NATNA is complementary to the second DNAtarget sequence. Furthermore, the Cpf1 protein of the bound nucleicacid/protein composition is capable of cutting the first DNA targetsequence and the Cas9 protein of the bound nucleic acid/proteincomposition is capable of cutting the second DNA target sequence.

These aspects and other embodiments of the present invention using theengineered Class 2 CRISPR-Cas systems of the present invention will bereadily apparent to those of ordinary skill in the art in view of thedisclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

The figures are not proportionally rendered, nor are they to scale. Thelocations of indicators are approximate.

FIG. 1A and FIG. 1B present illustrative examples of dual-guide Class 2Type II CRISPR-associated guide RNAs.

FIG. 2 presents an illustrative example of single-guide Class 2 Type IICRISPR-associated guide RNA.

FIG. 3 presents an illustrative example of a Class 2 Type V crRNA guideRNA.

FIG. 4, FIG. 5A, FIG. 5B, FIG. 6, FIG. 7, FIG. 8, FIG. 9, and FIG. 10present examples of engineered Cas9-Cpf1 nucleic-acid targeting nucleicacids of the present invention.

FIG. 11A, FIG. 11B, FIG. 11C, and FIG. 11D illustrate excision of asequence from a nucleic acid target sequence using an engineeredCas9-Cpf1 nucleic-acid targeting nucleic acid and Cas9 and Cpf1 proteinsboth having active endonucleases.

FIG. 12A, FIG. 12B, and FIG. 12C illustrate using an engineeredCas9-Cpf1 nucleic-acid targeting nucleic acid and Cas9 and Cpf1proteins, wherein endonuclease domains of Cas9 are active and theendonuclease domain of Cpf1 is inactive, to bring a donor polynucleotideinto proximity of a DSB in a nucleic acid target sequence.

FIG. 13A, FIG. 13B, and FIG. 13C illustrate using an engineeredCas9-Cpf1 nucleic-acid targeting nucleic acid and Cas9 and Cpf1proteins, wherein endonuclease domains of Cas9 are inactive and theendonuclease domain of Cpf1 is active, to bring a donor polynucleotideinto proximity of a double-strand break in a nucleic acid targetsequence.

FIG. 14A through FIG. 14E depict Cpf1-crRNAs. FIG. 14A illustrates awild-type Cpf1-crRNA. FIG. 14B illustrates a wild-type Cpf1 pre-crRNA.FIG. 14C, FIG. 14D and FIG. 14E illustrate modified Cpf1-crRNAs.

FIG. 15 presents the results of F. novicida Cpf1 biochemical cleavageassays using various modified Cpf1 crRNAs.

INCORPORATION BY REFERENCE

All patents, publications, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpatent, publication, or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a,” “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a polynucleotide” includes one or more polynucleotides, and referenceto “a vector” includes one or more vectors.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although other methods andmaterials similar, or equivalent, to those described herein can beuseful in the present invention, preferred materials and methods aredescribed herein.

In view of the teachings of the present specification, one of ordinaryskill in the art can employ conventional techniques of immunology,biochemistry, chemistry, molecular biology, microbiology, cell biology,genomics, and recombinant polynucleotides, as taught, for example, bythe following standard texts: Antibodies: A Laboratory Manual, Secondedition, E. A. Greenfield, Cold Spring Harbor Laboratory Press, ISBN978-1-936113-81-1 (2014); Culture of Animal Cells: A Manual of BasicTechnique and Specialized Applications, 6th Edition, R. I. Freshney,Wiley-Blackwell, ISBN 978-0-470-52812-9 (2010); Transgenic AnimalTechnology, Third Edition: A Laboratory Handbook, C. A. Pinkert,Elsevier, ISBN 978-0124104907 (2014); The Laboratory Mouse, SecondEdition, H. Hedrich, Academic Press, ISBN 978-0123820082 (2012);Manipulating the Mouse Embryo: A Laboratory Manual, R. Behringer, etal., Cold Spring Harbor Laboratory Press, ISBN 978-1936113019 (2013);PCR 2: A Practical Approach, M. J. McPherson, et al., IRL Press, ISBN978-0199634248 (1995); Methods in Molecular Biology (Series), J. M.Walker, ISSN 1064-3745, Humana Press; RNA: A Laboratory Manual, D. C.Rio , et al., Cold Spring Harbor Laboratory Press, ISBN 978-0879698911(2010); Methods in Enzymology (Series), Academic Press; MolecularCloning: A Laboratory Manual (Fourth Edition), M. R. Green, et al., ColdSpring Harbor Laboratory Press, ISBN 978-1605500560 (2012); BioconjugateTechniques, Third Edition, G. T. Hermanson, Academic Press, ISBN978-0123822390 (2013); Methods in Plant Biochemistry and MolecularBiology, W. V. Dashek, CRC Press, ISBN 978-0849394805 (1997); Plant CellCulture Protocols (Methods in Molecular Biology), V. M. Loyola-Vargas,et al., Humana Press, ISBN 978-1617798177 (2012); Plant TransformationTechnologies, C. N. Stewart, et al., Wiley-Blackwell, ISBN978-0813821955 (2011); Recombinant Proteins from Plants (Methods inBiotechnology), C. Cunningham, et al., Humana Press, ISBN 978-1617370212(2010); Plant Genomics: Methods and Protocols (Methods in MolecularBiology), D. J. Somers, et al., Humana Press, ISBN 978-1588299970(2009); Plant Biotechnology: Methods in Tissue Culture and GeneTransfer, R. Keshavachandran, et al., Orient Blackswan, ISBN978-8173716164 (2008).

Clustered regularly interspaced short palindromic repeats (CRISPR) andrelated CRISPR-associated proteins (Cas proteins) constitute CRISPR-Cassystems (see, e.g., Barrangou, R., et al., Science 315:1709-1712(2007)).

As used herein, “Cas protein” and “CRISPR-Cas protein” refer toCRISPR-associated proteins (Cas) including, but not limited to Class 1Type I CRISPR-associated proteins, Class 1 Type III CRISPR-associatedproteins, and Class 1 Type IV CRISPR-associated proteins, Class 2 TypeII CRISPR-associated proteins, Class 2 Type V CRISPR-associatedproteins, and Class 2 Type VI CRISPR-associated proteins. Class 2 Casproteins include Cas9 proteins, Cas9-like proteins encoded by Cas9orthologs, Cas9-like synthetic proteins, Cpf1 proteins, proteins encodedby Cpf1 orthologs, Cpf1-like synthetic proteins, C2c1 proteins, C2c2proteins, C2c3 proteins, and variants and modifications thereof. In someembodiments, Cas proteins are Class 2 CRISPR-associated proteins, forexample one or more Class 2 Type II CRISPR-associated proteins, such asCas9, one or more Class 2 Type V CRISPR-associated proteins, such asCpf1, and one ore more Class 2 Type VI CRISPR-associated proteins, suchas C2c2. In preferred embodiments, Cas proteins are one or more Class 2Type II CRISPR-associated proteins, such as Cas9, and one or more Class2 Type V CRISPR-associated proteins, such as Cpf1. Typically, for use inaspects of the present invention, a Cas protein is capable ofinteracting with one or more cognate polynucleotides (most typicallyRNA) to form a nucleoprotein complex (most typically, aribonucleoprotein complex).

“Cas9 protein,” as used herein, refers to a Cas9 wild-type proteinderived from Class 2 Type II CRISPR-Cas9 systems, modifications of Cas9proteins, variants of Cas9 proteins, Cas9 orthologs, and combinationsthereof. Cas9 proteins include, but not limited to, Cas9 fromStreptococcus pyogenes (UniProtKB-Q99ZW2 (CAS9_STRP1)), Streptococcusthermophilus (UniProtKB-G3ECR1 (CAS9_STRTR)), and Staphylococcus aureus(UniProtKB-J7RUA5 (CAS9_STAAU)). Cas9 homologs can be identified usingsequence similarity search methods known to one skilled in the art.“dCas9,” as used herein, refers to variants of Cas9 protein that arenuclease-deactivated Cas9 proteins, also termed “catalytically inactiveCas9 protein,” “enzymatically inactive Cas9,” “catalytically dead Cas9”or “dead Cas9.” Such molecules lack all or a portion of endonucleaseactivity and can therefore be used to regulate genes in an RNA-guidedmanner (see Jinek M., et al., Science 337:816-821 (2012)). This isaccomplished by introducing mutations that inactivate Cas9 nucleasefunction and is typically accomplished by mutating both of the twocatalytic residues (D10A in the RuvC-1 domain, and H840A in the HNHdomain, numbered relative to S. pyogenes Cas9). It is understood thatmutation of other catalytic residues to reduce activity of either orboth of the nuclease domains can also be carried out by one skilled inthe art. The resultant dCas9 is unable to cleave double-stranded DNA butretains the ability to complex with a guide nucleic acid and bind a DNAtarget sequence. The Cas9 double mutant with changes at amino acidpositions D10A and H840A completely inactivates both the nuclease andnickase activities. Targeting specificity is determined by complementarybase pairing of guide RNA (typically, a single guide RNA) to the genomiclocus and the PAM. Cas9 is the signature protein characteristic forClass 2 Type II CRISPR systems.

“Cpf1 protein,” as used herein, refers to a Cpf1 wild-type proteinderived from Class 2 Type V CRISPR-Cpf1 systems, modifications of Cpf1proteins, variants of Cpf1 proteins, Cpf1 orthologs, and combinationsthereof. “dCpf1, ” as used herein, refers to variants of Cpf1 proteinthat are nuclease-deactivated Cpf1 proteins, also termed “catalyticallyinactive Cpf1 protein,” or “enzymatically inactive Cpf1.” Cpf1 proteinsinclude, but not limited to, Francisella novicida (UniProtKB-A0Q7Q2(CPF1_FRATN)), Lachnospiraceae bacterium (UniProtKB-A0A182DWE3(A0A182DWE3_9FIRM)), and Acidaminococcus sp. (UniProtKB-U2UMQ6(CPF1_ACISB)). Cpf1 is the signature protein characteristic for Class 2Type V CRISPR systems. Cpf1 homologs can be identified using sequencesimilarity search methods known to one skilled in the art.

“Argonaute protein,” as used herein, refers to an Argonaute wild-typeprotein, modifications of Argonaute proteins, variants of Argonauteproteins, Argonaute orthologs, and combinations thereof (see, e.g.,Hall, T., Structure 13:1403-1408 (2005); Hock, J., et al., GenomeBiology 9(2): 210-210.8 (2008); Swarts, D., et al., Nature507(7491):258-261 (2014); Swarts, D., et al., Nature Structural &Molecular Biology (9):743-753 (2014); Hur, J. K., et al., Trends inBiochemical Sciences 39(6):257-259 (2014)). “dArgonaute,” as usedherein, refers to variants of Argonaute proteins that arenuclease-deactivated Argonaute proteins, also termed “catalyticallyinactive Argonaute protein,” or “enzymatically inactive Argonaute.”Argonaute protein refers to a protein from a family of proteinstypically defined by the presence of a PIWI domain and/or a PAZ(PIWI-Argonaute-Zwille) domain. An Argonaute protein (e.g., a eukaryoticArgonaute or prokaryotic Argonaute) typically is capable of interactingwith an Argonaute guide (i.e., a nucleic-acid targeting nucleic acid;Ago-NATNA) to form a complex. The Ago-NATNA comprises a nucleic acidtarget binding sequence. The complex is capable of site-directed bindingto a nucleic acid target sequence. The complex is targeted to thenucleic acid target sequence by the nucleic acid target binding sequenceof the Ago-NATNA. The nucleic acid target sequences to which anArgonaute protein/Ago-NATNA complex binds can be, for example, RNA, DNA,or hybrids of RNA/DNA. Argonaute protein homologs can be identifiedusing sequence similarity search methods known to one skilled in theart.

By “nucleic-acid targeting nucleic acid” (NATNA) is meant one or morepolynucleotides that guide a protein, such as an Argonaute protein ormore preferably a Cas protein (e.g., a Cas9 protein, a dCas9 protein, aCpf1 protein, or a dCpf1 protein) to preferentially bind a nucleic acidtarget sequence, typically, a double-stranded nucleic acid targetsequence. For example, embodiments of the present invention include, butare not limited to, engineered NATNAs comprising a Cas9-associatednucleic-acid targeting nucleic acid and a Cpf1-associated nucleic-acidtargeting nucleic acid. Examples of these NATNAs include, but are notlimited to the following: a Cas9-associated nucleic-acid targetingnucleic acid comprising a spacer element (“first Cas9-NATNA”; e.g., FIG.1A, 101) and a Cas9-associated nucleic-acid targeting nucleic acidcomprising a tracr element (“second Cas9-NATNA”; e.g., FIG. 1A, 102); a“single-Cas9-NATNA” (see, e.g., FIG. 2) comprising a first Cas9-NATNAand a second Cas9-NATNA; and a Cpf1-associated nucleic-acid targetingnucleic acid comprising a spacer element (“Cpf1-NATNA”) (see, e.g., FIG.3). Examples of Cas9-Cpf1-NATNAs of one aspect of the present inventioninclude, but are not limited to, those illustrated in FIG. 3, FIG. 4,FIG. 5A, FIG. 5B, FIG. 6, FIG. 7, FIG. 8, FIG. 9, and FIG. 10. NATNAscan comprise ribonucleotide bases (e.g., RNA), deoxyribonucleotide bases(e.g., DNA), combinations of ribonucleotide bases anddeoxyribonucleotide bases (e.g., RNA/DNA), nucleotides, nucleotideanalogs, modified nucleotides, and the like, as well as synthetic,naturally occurring, and non-naturally occurring modified backboneresidues or linkages, for example, as described herein.

As used herein, “dual-guide RNA” and “Cas9-dual-guide RNA” typicallyrefer to a two-component RNA system for a polynucleotide componentcapable of associating with a cognate Cas9 protein. FIG. 1A and FIG. 1Bpresent illustrative examples of dual-guide Class 2 Type IICRISPR-Cas9-associated RNAs. FIG. 1A shows a two-RNA component Type IICRISPR-Cas9 system comprising a Cas9-crRNA (FIG. 1A, 101) and aCas9-tracrRNA (FIG. 1A, 102). FIG. 1B illustrates the formation ofbase-pair hydrogen bonds between the Cas9-crRNA and the Cas9-tracrRNA toform secondary structure (see U.S. Published Patent Application No.2014-0068797, published 6 Mar. 2014; see also Jinek M., et al., Science337:816-21 (2012)). FIG. 1B presents an overview of and nomenclature forsecondary structural elements of the Cas9-crRNA and Cas9-tracrRNA of theS. pyogenes Cas9 including the following: a spacer element (FIG. 1B,103); a first stem element comprising a lower stem element (FIG. 1B,104), a bulge element comprising unpaired nucleotides (FIG. 1B, 105),and an upper stem element (FIG. 1B, 106); a nexus element (FIG. 1B,107); a first 3′ hairpin element (FIG. 1B, 108); and a second 3′ hairpinelement (FIG. 1B, 109). A Cas9-dual-guide RNA is capable of forming anucleoprotein complex with a cognate Cas9 protein, wherein the complexis capable of targeting a nucleic acid target sequence complementary tothe spacer sequence. Modifications of Cas9-dual guides are known in theart, including, deletion of one or more 3′ hairpin elements (FIG. 1B,108, 109) and modifications of the upper stem, bulge, and lower stem(FIG. 1B, 106, 105, 104, respectively) (see, e.g., U.S. PatentPublication No. 2014-0315985, published 23 Oct. 2014; U.S. PatentPublication No. 2015-0376586, published 31 Dec. 2015).

As used herein, “single-guide RNA” (sgRNA) and “Cas9-sgRNA” typicallyrefer to a one-component RNA system for a polynucleotide componentcapable of associating with a cognate Cas9 protein. FIG. 2 shows anexample of a Class 2 Type II CRISPR-Cas9-associated sgRNA. The figureillustrates a Cas9 single-guide RNA (Cas9-sgRNA) wherein the Cas9-crRNAis covalently joined to the Cas9-tracrRNA and forms a RNA polynucleotidesecondary structure through base-pair hydrogen bonding (see, e.g., U.S.Published Patent Application No. 2014-0068797, published 6 Mar. 2014).FIG. 2 presents an overview of and nomenclature for secondary structuralelements of a Cas9-sgRNA for S. pyogenes including the following: aspacer element (FIG. 2, 201); a first stem element comprising a lowerstem element (FIG. 2, 202), a bulge element comprising unpairednucleotides (FIG. 2, 205), and an upper stem element (FIG. 2, 203); aloop element (FIG. 2, 204) comprising unpaired nucleotides; a nexuselement (FIG. 2, 206); a first 3′ hairpin element (FIG. 2, 207); and asecond 3′ hairpin element (FIG. 2, 208). (See, e.g., FIGS. 1 and 3 ofBriner, A. E., et al., Molecular Cell 56(2):333-339 (2014).) ACas9-sgRNA is capable of forming a nucleoprotein complex with a cognateCas9 protein, wherein the complex is capable of targeting a nucleic acidsequence complementary to the spacer sequence. Modifications ofCas9-single guides are known in the art, including, deletion of one ormore 3′ hairpin elements (FIG. 2, 207, 208) and modifications of theupper stem, bulge, and lower stem (FIG. 2, 203, 205, 202, respectively)(see, e.g., U.S. Patent Publication No. 2014-0315985, published 23 Oct.2014; U.S. Patent Publication No. 2015-0376586, published 31 Dec. 2015).

“Guide crRNA” and “Cpf1-crRNA,” as used herein, typically refer to aone-component RNA system for a polynucleotide component capable ofassociating with a cognate Cpf1 protein. FIG. 3 presents an example of aType V CRISPR-Cpf1-associated RNA (Cpf1-crRNA) (see, e.g., Zetsche, B.,et al., Cell 163:1-13 (2015)). FIG. 3 presents an overview of andnomenclature for secondary structural elements of a Cpf1-crRNA asfollows: a stem-loop element (FIG. 3, 301) and a spacer element (FIG. 3,302). The stem-loop element comprises, in a 5′ to 3′ direction, aCpf1-stem RNA sequence 1 (FIG. 3, 303), a loop element (FIG. 3, 304),and a complementary Cpf1-stem RNA sequence 2 (FIG. 3, 305), wherein theCpf1-stem RNA sequence 1 and the complementary Cpf1-stem RNA sequence 2form a duplex. A guide crRNA is capable of forming a nucleoproteincomplex with a cognate Cpf1 protein, wherein the complex is capable oftargeting a nucleic acid target sequence complementary to the spacersequence.

As used herein, a “cross-type-nucleic-acid targeting nucleic acid” and a“cross-type-NATNA” are used interchangeably to refer to a first NATNAconnected with a second NATNA. For example, a CRISPR Class 2cross-type-NATNA refers to one or more polynucleotides typicallycomprising a CRISPR Class 2 Type V NATNA (e.g., FIG. 3) connected with aCRISPR Class 2 Type II NATNA (e.g., FIG. 1A, FIG. 1B, FIG. 2). The typesof connections, between the first NATNA and the second NATNA, to form across-type-NATNA include, for example, covalent linkage (e.g., FIG. 4),hydrogen bonding (e.g., FIG. 5A, FIG. 5B), ligand/ligand binding moietypairing, and/or cross-linking. Covalent linkages include, but are notlimited, to phosphodiester bonds. A cross-type-NATNA is capable offorming a complex with a first protein that is capable of forming acomplex with the first NATNA and a second protein that is capable offorming a complex with the second NATNA. For example, a CRISPR Class 2cross-type-NATNA is capable of forming a complex with a Class 2 Type VCas protein (e.g., a Cpf1 protein) that is capable of forming a complexwith the CRISPR Class 2 Type V NATNA and Class 2 Type II Cas protein(e.g., a Cas9 protein) that is capable of forming a complex with theCRISPR Class 2 Type II NATNA. In this embodiment, a complex formedbetween the Cpf1-NATNA and a Cpf1 protein is capable of binding a firstdouble-stranded nucleic acid target sequence. Also, and a complex formedbetween the Cas9-NATNA (e.g., a first Cas9-associated nucleic-acidtargeting nucleic acid comprising a spacer element, “first Cas9-NATNA,”and a second Cas9-associated nucleic-acid targeting nucleic acidcomprising a tracr element, “second Cas9-NATNA, or a single-Cas9-NATNA)and a Cas9 protein is capable of binding a second double-strandednucleic acid target sequence (e.g., FIG. 11B). In another embodiment, across-type-NATNA comprises an Ago-NATNA and a Class 2 Type V NATNA or aClass 2 Type II NATNA.

As used herein, a “cross-link” is a bond that links one polymer chain(e.g., a polynucleotide or polypeptide) to another. Such bonds can becovalent bonds or ionic bonds. For example, one polynucleotide can bebound to another polynucleotide by cross-linking the polynucleotides.

As used herein, the term “cognate” typically refers to a Cas protein andone or more Cas polynucleotides that are capable of forming anucleoprotein complex capable of site-directed binding to a nucleic acidtarget sequence complementary to the nucleic acid target bindingsequence present in one of the Cas polynucleotides.

As used herein, “complementarity” refers to the ability of a nucleicacid sequence to form hydrogen bond(s) with another nucleic acidsequence (e.g., through traditional Watson-Crick base pairing). Apercent complementarity indicates the percentage of residues in anucleic acid molecule that can form hydrogen bonds with a second nucleicacid sequence. If two polynucleotide sequences have 100%complementarity, the two sequences are perfectly complementary, i.e.,all of the contiguous residues of a first polynucleotide hydrogen bondwith the same number of contiguous residues in a second polynucleotide.

“Covalent bond,” “covalently attached,” “covalently bound,” “covalentlylinked,” “covalently connected,” and “molecular bond” are usedinterchangeably herein, and refer to a chemical bond that involves thesharing of electron pairs between atoms. Examples of covalent bondsinclude, but are not limited to, phosphodiester bonds andphosphorothioate bonds.

“Non-covalent bond,” “non-covalently attached,” “non-covalently bound,”“non-covalently linked,” “non-covalent interaction,” and “non-covalentlyconnected” are used interchangeably herein, and refer to any relativelyweak chemical bond that does not involve sharing of a pair of electrons.Multiple non-covalent bonds often stabilize the conformation ofmacromolecules and mediate specific interactions between molecules.Examples of non-covalent bonds include, but are not limited to hydrogenbonding (e.g., a Watson-Crick-type hydrogen-bonded base pair, and aHoogsteen base pair), ionic interactions (e.g., NaCl), van der Waalsinteractions, and hydrophobic bonds.

“Connect,” “connected,” and “connecting” are used interchangeablyherein, and refer to a covalent bond or a non-covalent bond between twomacromolecules (e.g., polynucleotides, proteins, and the like).

As used herein, “binding” refers to a non-covalent interaction betweenmacromolecules (e.g., between a protein and a polynucleotide, between apolynucleotide and a polynucleotide, and between a protein and aprotein, and the like). Such non-covalent interaction is also referredto as “associating” or “interacting” (e.g., if a first macromoleculeinteracts with a second macromolecule, the first macromolecule binds tosecond macromolecule in a non-covalent manner). Some portions of abinding interaction may be sequence-specific (the terms“sequence-specific binding,” “sequence-specifically bind,”“site-specific binding,” and “site-specifically binds” are usedinterchangeably herein). Sequence-specific binding, as used herein,typically refers to one or more NATNAs capable of forming a complex witha protein (e.g., a Cas protein or an Argonaute protein) to cause theprotein to bind a first nucleic acid sequence (e.g., a first DNAsequence) comprising a first nucleic acid target sequence (e.g., a firstDNA target sequence) preferentially relative to a second nucleic acidsequence (e.g., a second DNA sequence) without the nucleic acid targetbinding sequence (e.g., the first DNA target binding sequence). Allcomponents of a binding interaction do not need to be sequence-specific,such as contacts of a protein with phosphate residues in a DNA backbone.Binding interactions can be characterized by a dissociation constant(Kd). “Affinity” refers to the strength of binding. An increased bindingaffinity is correlated with a lower Kd.

As used herein, a Cas protein (e.g., a Cas9 protein or Cpf1 protein) issaid to “target” a polynucleotide if a Cas protein/NATNA nucleoproteincomplex binds or cleaves a polynucleotide at the nucleic acid targetsequence within the polynucleotide. An Argonaute protein is said totarget a polynucleotide if an Argonaute protein/NATNA nucleoproteincomplex binds or cleaves a polynucleotide at the nucleic acid targetsequence within the polynucleotide.

As used herein, “double-strand break” (DSB) refers to both strands of adouble-stranded segment of DNA being severed. In some instances, if sucha break occurs, one strand can be said to have a “sticky end” whereinnucleotides are exposed and not hydrogen bonded to nucleotides on theother strand. In other instances, a “blunt end” can occur wherein bothstrands remain fully base paired with each other despite the DSB.

“Donor polynucleotide,” “donor oligonucleotide,” and “donor template”are used interchangeably herein and can be a double-strandpolynucleotide (e.g., DNA), a single-stranded polynucleotide (e.g., DNAoligonucleotides), or a combination thereof. Donor polynucleotidescomprise homology arms flanking the insertion sequence (e.g., DSBs inthe DNA). The homology arms on each side can vary in length. Parametersfor the design and construction of donor polynucleotides are well-knownin the art (see, e.g., Ran, F., et al., Nature Protocols 8(11):2281-2308(2013); Smithies, O., et al., Nature 317:230-234 (1985); Thomas, K., etal., Cell 44:419-428 (1986); Wu, S., et al., Nature Protocols3:1056-1076 (2008); Singer, B., et al., Cell 31:25-33 (1982); Shen, P.,et al., Genetics 112:441-457 (1986); Watt, V., et al., Proceedings ofthe National Academy of Sciences of the United States of America82:4768-4772 (1985), Sugawara, N., et al., Journal of Molecular CellBiology 12(2):563-575 (1992); Rubnitz, J., et al., Journal of MolecularCell Biology 4(11):2253-2258 (1984); Ayares, D., et al., Proceedings ofthe National Academy of Sciences of the United States of America83(14):5199-5203 (1986); Liskay, R, et al., Genetics 115(1):161-167(1987)).

As used herein, “homology-directed repair” (HDR) refers to DNA repairthat takes place in cells, for example, during repair of a DSB in DNA.HDR requires nucleotide sequence homology and uses a donorpolynucleotide to repair the sequence wherein the DSB (e.g., within aDNA target sequence) occurred. The donor polynucleotide generally hasthe requisite sequence homology with the sequence flanking the DSB sothat the donor polynucleotide can serve as a suitable template forrepair. HDR results in the transfer of genetic information from, forexample, the donor polynucleotide to the DNA target sequence. HDR mayresult in alteration of the DNA target sequence (e.g., insertion,deletion, mutation) if the donor polynucleotide sequence differs fromthe DNA target sequence and part or all of the donor polynucleotide isincorporated into the DNA target sequence. In some embodiments, anentire donor polynucleotide, a portion of the donor polynucleotide, or acopy of the donor polynucleotide is integrated at the site of the DNAtarget sequence. For example, a donor polynucleotide can be used forrepair of the break in the DNA target sequence, wherein the repairresults in the transfer of genetic information (i.e., polynucleotidesequences) from the donor polynucleotide at the site or in closeproximity of the break in the DNA. Accordingly, new genetic information(i.e., polynucleotide sequences) may be inserted or copied at a DNAtarget sequence.

A “genomic region” is a segment of a chromosome in the genome of a hostcell that is present on either side of the nucleic acid target sequencesite or, alternatively, also includes a portion of the nucleic acidtarget sequence site. The homology arms of the donor polynucleotide havesufficient homology to undergo homologous recombination with thecorresponding genomic regions. In some embodiments, the homology arms ofthe donor polynucleotide share significant sequence homology to thegenomic region immediately flanking the nucleic acid target sequencesite; it is recognized that the homology arms can be designed to havesufficient homology to genomic regions farther from the nucleic acidtarget sequence site.

As used herein, “non-homologous end joining” (NHEJ) refers to the repairof a DSB in DNA by direct ligation of one end of the break to the otherend of the break without a requirement for a donor polynucleotide. NHEJis a DNA repair pathway available to cells to repair DNA without the useof a repair template. NHEJ in the absence of a donor polynucleotideoften results in nucleotides being randomly inserted or deleted at thesite of the DSB.

“Microhomology-mediated end joining” (MMEJ) is pathway for repairing aDSB in DNA. MMEJ involves deletions flanking a DSB and alignment ofmicrohomologous sequences internal to the broken ends before joining.MMEJ is genetically defined and requires the activity of, for example,CtIP, Poly(ADP-Ribose) Polymerase 1 (PARP1), DNA polymerase theta (Polθ), DNA Ligase 1 (Lig 1), DNA Ligase 3 (Lig 3). Additional geneticcomponents are known in the art (see, e.g., Sfeir, A., et al., Trends inBiochemical Sciences 40:701-714 (2015)).

As used herein, “DNA repair” encompasses any process whereby cellularmachinery repairs damage to a DNA molecule contained in the cell. Thedamage repaired can include single-strand breaks or double-strandbreaks. At least three mechanisms exist to repair DSBs: HDR, NHEJ, andMMEJ. “DNA repair” is also used herein to refer to DNA repair resultingfrom human manipulation, wherein a target locus is modified, e.g., byinserting, deleting, substituting nucleotides, all of which representforms of genome editing.

As used herein, “recombination” refers to a process of exchange ofgenetic information between two polynucleotides.

As used herein, the terms “regulatory sequences,” “regulatory elements,”and “control elements” are interchangeable and refer to polynucleotidesequences that are upstream (5′ non-coding sequences), within, ordownstream (3′ non-translated sequences) of a polynucleotide target tobe expressed. Regulatory sequences influence, for example, the timing oftranscription, amount or level of transcription, RNA processing orstability, and/or translation of the related structural nucleotidesequence. Regulatory sequences may include activator binding sequences,enhancers, introns, polyadenylation recognition sequences, promoters,transcription start sites, repressor binding sequences, stem-loopstructures, translational initiation sequences, internal ribosome entrysites (IRES), translation leader sequences, transcription terminationsequences (e.g., polyadenylation signals and poly-U sequences),translation termination sequences, primer binding sites, and the like.

Regulatory elements include those that direct constitutive, inducible,and repressible expression of a nucleotide sequence in many types ofhost cells and those that direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).In some embodiments, a vector comprises one or more pol III promoters,one or more pol II promoters, one or more pol I promoters, orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer; see, e.g., Boshart et al.,Cell 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductasepromoter, the (3-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EFla promoter. It will be appreciated by those skilledin the art that the design of an expression vector can depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression desired, and the like. A vector can be introduced into hostcells to thereby produce transcripts, proteins, or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein.

“Gene,” as used herein, refers to a polynucleotide sequence comprisingexon(s) and related regulatory sequences. A gene may further compriseintron(s) and/or untranslated region(s) (UTR).

As used herein, the term “operably linked” refers to polynucleotidesequences or amino acid sequences placed into a functional relationshipwith one another. For example, regulatory sequences (e.g., a promoter orenhancer) are “operably linked” to a polynucleotide encoding a geneproduct if the regulatory sequences regulate or contribute to themodulation of the transcription of the polynucleotide. Operably linkedregulatory elements are typically contiguous with the coding sequence.However, enhancers can function if separated from a promoter by up toseveral kilobases or more. Accordingly, some regulatory elements may beoperably linked to a polynucleotide sequence but not contiguous with thepolynucleotide sequence. Similarly, translational regulatory elementscontribute to the modulation of protein expression from apolynucleotide.

As used herein, “expression” refers to transcription of a polynucleotidefrom a DNA template, resulting in, for example, a messenger RNA (mRNA)or other RNA transcript (e.g., non-coding, such as structural orscaffolding RNAs). The term further refers to the process through whichtranscribed mRNA is translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be referred to collectively as“gene product(s).” Expression may include splicing the mRNA in aeukaryotic cell, if the polynucleotide is derived from genomic DNA.

As used herein, the term “modulate” refers to a change in the quantity,degree or amount of a function. For example, a Cas9 protein/Cpf1protein/Cas9-Cpf1-NATNA complex, as disclosed herein, may modulate theactivity of a promoter sequence by binding to two nucleic acid targetsequences at or near the promoter. Depending on the action occurringafter binding, the Cas9 protein/Cpf1 protein/Cas9-Cpf1-NATNA complex caninduce, enhance, suppress, or inhibit transcription of a geneoperatively linked to the promoter sequence. Thus, “modulation” of geneexpression includes both gene activation and gene repression.

Modulation can be assayed by determining any characteristic directly orindirectly affected by the expression of the target gene. Suchcharacteristics include, e.g., changes in RNA or protein levels, proteinactivity, product levels, expression of the gene, or activity level ofreporter genes. Accordingly, the terms “modulating expression,”“inhibiting expression,” and “activating expression” of a gene can referto the ability of a Cas9 protein/Cpf1 protein/Cas9-Cpf1-NATNA complex tochange, activate, or inhibit transcription of a gene.

“Vector” and “plasmid,” as used herein, refer to a polynucleotidevehicle to introduce genetic material into a cell. Vectors can be linearor circular. Vectors can contain a replication sequence capable ofeffecting replication of the vector in a suitable host cell (i.e., anorigin of replication). Upon transformation of a suitable host, thevector can replicate and function independently of the host genome orintegrate into the host genome. Vector design depends, among otherthings, on the intended use and host cell for the vector, and the designof a vector of the invention for a particular use and host cell iswithin the level of skill in the art. The four major types of vectorsare plasmids, viral vectors, cosmids, and artificial chromosomes.Typically, vectors comprise an origin of replication, a multicloningsite, and/or a selectable marker. An expression vector typicallycomprises an expression cassette.

As used herein, “expression cassette” refers to a polynucleotideconstruct generated using recombinant methods or by synthetic means andcomprising regulatory sequences operably linked to a selectedpolynucleotide to facilitate expression of the selected polynucleotidein a host cell. For example, the regulatory sequences can facilitatetranscription of the selected polynucleotide in a host cell, ortranscription and translation of the selected polynucleotide in a hostcell. An expression cassette can, for example, be integrated in thegenome of a host cell or be present in a vector to form an expressionvector.

As used herein, a “targeting vector” is a recombinant DNA constructtypically comprising tailored DNA arms, homologous to genomic DNA, thatflank elements of a target gene or nucleic acid target sequence (e.g., aDSB). A targeting vector comprises a donor polynucleotide. Elements ofthe target gene can be modified in a number of ways including deletionsand/or insertions. A defective target gene can be replaced by afunctional target gene, or in the alternative a functional gene can beknocked out. Optionally, the donor polynucleotide of a targeting vectorcomprises a selection cassette comprising a selectable marker that isintroduced into the target gene. Targeting regions (i.e., nucleic acidtarget sequences) adjacent or within a target gene can be used to affectregulation of gene expression.

As used herein, the terms “nucleic acid,” “nucleotide sequence,”“oligonucleotide,” and “polynucleotide” are interchangeable and refer toa polymeric form of nucleotides. The nucleotides may bedeoxyribonucleotides (DNA), ribonucleotides (RNA), analogs thereof, orcombinations thereof, and may be of any length. Polynucleotides mayperform any function and may have any secondary and tertiary structures.The terms encompass known analogs of natural nucleotides and nucleotidesthat are modified in the base, sugar and/or phosphate moieties. Analogsof a particular nucleotide have the same base-pairing specificity (e.g.,an analog of A base pairs with T). A polynucleotide may comprise onemodified nucleotide or multiple modified nucleotides. Examples ofmodified nucleotides include fluorinated nucleotides, methylatednucleotides, and nucleotide analogs. Nucleotide structure may bemodified before or after a polymer is assembled. Followingpolymerization, polynucleotides may be additionally modified via, forexample, conjugation with a labeling component or target bindingcomponent. A nucleotide sequence may incorporate non-nucleotidecomponents. The terms also encompass nucleic acids comprising modifiedbackbone residues or linkages, that are synthetic, naturally occurring,and non-naturally occurring, and have similar binding properties as areference polynucleotide (e.g., DNA or RNA). Examples of such analogsinclude, but are not limited to, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), Locked Nucleic Acid(LNA™) (Exiqon, Inc., Woburn, Mass.) nucleosides, glycol nucleic acid,bridged nucleic acids, and morpholino structures.

Peptide-nucleic acids (PNAs) are synthetic homologs of nucleic acidswherein the polynucleotide phosphate-sugar backbone is replaced by aflexible pseudo-peptide polymer. Nucleobases are linked to the polymer.PNAs have the capacity to hybridize with high affinity and specificityto complementary sequences of RNA and DNA.

In phosphorothioate nucleic acids, the phosphorothioate (PS) bondsubstitutes a sulfur atom for a non-bridging oxygen in thepolynucleotide phosphate backbone. This modification makes theinternucleotide linkage resistant to nuclease degradation. In someembodiments, phosphorothioate bonds are introduced between the last 3 to5 nucleotides at the 5′ or 3′ end of a polynucleotide sequence toinhibit exonuclease degradation. Placement of phosphorothioate bondsthroughout an entire oligonucleotide helps reduce degradation byendonucleases as well.

Threose nucleic acid (TNA) is an artificial genetic polymer. Thebackbone structure of TNA comprises repeating threose sugars linked byphosphodiester bonds. TNA polymers are resistant to nucleasedegradation. TNA can self-assemble by base-pair hydrogen bonding intoduplex structures.

Linkage inversions can be introduced into polynucleotides through use of“reversed phosphoramidites” (see, e.g.,www.ucalgary.ca/dnalab/synthesis/-modifications/linkages). Typically,such polynucleotides have phosphoramidite groups on the 5′-OH positionand a dimethoxytrityl (DMT) protecting group on the 3′-OH position.Normally, the DMT protecting group is on the 5′-OH and thephosphoramidite is on the 3′-OH. The most common use of linkageinversion is to add a 3′-3′ linkage to the end of a polynucleotide witha phosphorothioate backbone. The 3′-3′ linkage stabilizes thepolynucleotide to exonuclease degradation by creating an oligonucleotidehaving two 5′-OH ends and no 3′-OH end.

Polynucleotide sequences are displayed herein in the conventional 5′ to3′ orientation unless otherwise indicated.

As used herein, “sequence identity” generally refers to the percentidentity of nucleotide bases or amino acids comparing a firstpolynucleotide or polypeptide to a second polynucleotide or polypeptideusing algorithms having various weighting parameters. Sequence identitybetween two polynucleotides or two polypeptides can be determined usingsequence alignment by various methods and computer programs (e.g.,BLAST, CS-BLAST, FASTA, HMMER, L-ALIGN, and the like) available throughthe worldwide web at sites including but not limited to GENBANK(www.ncbi.nlm.nih.gov/genbank/) and EMBL-EBI (www.ebi.ac.uk.). Sequenceidentity between two polynucleotides or two polypeptide sequences isgenerally calculated using the standard default parameters of thevarious methods or computer programs. A high degree of sequenceidentity, as used herein, between two polynucleotides or twopolypeptides is typically between about 90% identity and 100% identity,for example, about 90% identity or higher, preferably about 95% identityor higher, more preferably about 98% identity or higher. A moderatedegree of sequence identity, as used herein, between two polynucleotidesor two polypeptides is typically between about 80% identity to about 85%identity, for example, about 80% identity or higher, preferably about85% identity. A low degree of sequence identity, as used herein, betweentwo polynucleotides or two polypeptides is typically between about 50%identity and 75% identity, for example, about 50% identity, preferablyabout 60% identity, more preferably about 75% identity. For example, aCas protein (e.g., a Cas9 comprising amino acid substitutions or a Cpf1comprising amino acid substitutions) can have a moderate degree ofsequence identity, or preferably a high degree of sequence identity,over its length to a reference Cas protein (e.g., a wild-type Cas9 or awild-type Cpf1, respectively). As another example, a NATNA can have amoderate degree of sequence identity, or preferably a high degree ofsequence identity, over its length compared to a reference wild-typepolynucleotide that complexes with the reference Cas protein (e.g., ansgRNA that forms a complex with Cas9 or a crRNA that forms a complexwith Cpf1 ).

As used herein, “hybridization” or “hybridize” or “hybridizing” is theprocess of combining two complementary single-stranded DNA or RNAmolecules so as to form a single double-stranded molecule (DNA/DNA,DNA/RNA, RNA/RNA) through hydrogen base pairing. Hybridizationstringency is typically determined by the hybridization temperature andthe salt concentration of the hybridization buffer; e.g., hightemperature and low salt provide high stringency hybridizationconditions. Examples of salt concentration ranges and temperature rangesfor different hybridization conditions are as follows: high stringency,approximately 0.01M to approximately 0.05M salt, hybridizationtemperature 5° C. to 10° C. below T_(m); moderate stringency,approximately 0.16M to approximately 0.33M salt, hybridizationtemperature 20° C. to 29° C. below T_(m); and low stringency,approximately 0.33M to approximately 0.82M salt, hybridizationtemperature 40° C. to 48° C. below T_(m). T. of duplex nucleic acids iscalculated by standard methods well-known in the art (see, e.g.,Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press: New York (1982); Casey, J., et al.,Nucleic Acids Research 4:1539-1552 (1977); Bodkin, D. K., et al.,Journal of Virological Methods 10(1):45-52 (1985); Wallace, R. B., etal., Nucleic Acids Research 9(4):879-894 (1981)). Algorithm predictiontools to estimate T. are also widely available. High stringencyconditions for hybridization typically refer to conditions under which anucleic acid having complementarity to a target sequence predominantlyhybridizes with the target sequence, and substantially does nothybridize to non-target sequences. Typically, hybridization conditionsare of moderate stringency, preferably high stringency.

As used herein, a “stem-loop structure” or “stem-loop element” refers toa polynucleotide having a secondary structure that includes a region ofnucleotides that are known or predicted to form a double-stranded region(the “stem element”), wherein at one end of the double-stranded regioneach strand of the double-stranded region is linked by a region ofpredominantly single-stranded nucleotides (the “loop element”). The term“hairpin” element is also used herein to refer to stem-loop structures.Such structures are well known in the art. The base pairing may beexact; however, as is known in the art, a stem element does not requireexact base pairing. Thus, the stem element may include one or more basemismatches or non-paired bases.

A “linker element nucleotide sequence” and “linker nucleotide sequence”are used interchangeable herein and typically refer to a sequence of oneor more nucleotides covalently attached to a 5′ end, a 3′ end, or toboth the 5′ and 3′ ends of a first polynucleotide sequence. In someembodiments, the linker element nucleotide sequence is for the purposeof connecting a first polynucleotide to a second polynucleotide.

As used herein, the term “amino acid” refers to natural and synthetic(unnatural) amino acids, including amino acid analogs, modified aminoacids, peptidomimetics, glycine, and D or L optical isomers.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areinterchangeable and refer to polymers of amino acids. A polypeptide maybe of any length. It may be branched or linear, it may be interrupted bynon-amino acids, and it may comprise modified amino acids. The terms maybe used to refer to an amino acid polymer that has been modifiedthrough, for example, acetylation, disulfide bond formation,glycosylation, lipidation, phosphorylation, pegylation, biotinylation,cross-linking, and/or conjugation (e.g., with a labeling component orligand). Polypeptide sequences are displayed herein in the conventionalN-terminal to C-terminal orientation.

Polypeptides and polynucleotides can be made using routine techniques inthe field of molecular biology (see, e.g., standard texts discussedabove). Furthermore, essentially any polypeptide or polynucleotide isavailable from commercial sources.

The terms “fusion protein” and “chimeric protein,” as used herein, referto a single protein created by joining two or more proteins, proteindomains, or protein fragments that do not naturally occur together in asingle protein. For example, a fusion protein can contain a first domainfrom a Cas9 and a second domain from a Cpf1 protein, a first domain froma Cas9 or Cpf1 protein and a second domain from an Argonaute protein, afirst domain from a Cas9, Cpf1, or Argonaute protein and a second domainfrom a protein other than Cas9, Cpf1, or Argonaute protein, and so on.The modification to include such domains in fusion protein may conferadditional activity on the modified site-directed polypeptides. Suchactivities can include nuclease activity, methyltransferase activity,demethylase activity, DNA repair activity, DNA damage activity,deamination activity, dismutase activity, alkylation activity,depurination activity, oxidation activity, pyrimidine dimer formingactivity, integrase activity, transposase activity, recombinaseactivity, polymerase activity, ligase activity, helicase activity,photolyase activity, glycosylase activity, acetyltransferase activity,deacetylase activity, kinase activity, phosphatase activity, ubiquitinligase activity, deubiquitinating activity, adenylation activity,deadenylation activity, SUMOylating activity, deSUMOylating activity,ribosylation activity, deribosylation activity, myristoylation activityor demyristoylation activity) that modifies a polypeptide associatedwith nucleic acid target sequence (e.g., a histone). A fusion proteincan also comprise epitope tags (e.g., histidine tags, FLAG® (SigmaAldrich, St. Louis, Mo.) tags, Myc tags), reporter protein sequences(e.g., glutathione-S-transferase, beta-galactosidase, luciferase, greenfluorescent protein, cyan fluorescent protein, yellow fluorescentprotein), and/or nucleic acid binding domains (e.g., a DNA bindingdomain, an RNA binding domain). A fusion protein can also compriseactivator domains (e.g., heat shock transcription factors, NFKBactivators) or repressor domains (e.g., a KRAB domain). As described byLupo, A., et al., Current Genomics 14(4): 268-278 (2013), the KRABdomain is a potent transcriptional repression module and is located inthe amino-terminal sequence of most C2H2 zinc finger proteins (see,e.g., Margolin, J., et al., Proceedings of the National Academy ofSciences of the United States of America 91:4509-4513 (1994); Witzgall,R., et al., Proceedings of the National Academy of Sciences of theUnited States of America 91:4514-4518 (1994)). The KRAB domain typicallybinds to co-repressor proteins and/or transcription factors viaprotein-protein interactions, causing transcriptional repression ofgenes to which KRAB zinc finger proteins (KRAB-ZFPs) bind (see, e.g.,Friedman J R, et al., Genes & Development 10:2067-2678 (1996)). In someembodiments, linker nucleic acid sequences are used to join the two ormore proteins, protein domains, or protein fragments.

As used herein, a “host cell” generally refers to a biological cell. Acell is the basic structural, functional and/or biological unit of aliving organism. A cell can originate from any organism having one ormore cells. Examples of host cells include, but are not limited to: aprokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, acell of a single-cell eukaryotic organism, a protozoal cell, a cell froma plant (e.g., cells from plant crops, (such as soy, tomatoes, sugarbeets, pumpkin, hay, cannabis, tobacco, plantains, yams, sweet potatoes,cassava, potatoes, wheat, sorghum, soybean, rice, corn, maize,oil-producing Brassica (e.g., oil-producing rapeseed and canola),cotton, sugar cane, sunflower, millet, and alfalfa), fruits, vegetables,grains, seeds, flowering plants, conifers, gymnosperms, ferns,clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g.,Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsisgaditana, Chlorella pyrenoidosa, Sargassum patens C. agardh, and thelike), seaweeds (e.g., kelp), a fungal cell (e.g., a yeast cell, a cellfrom a mushroom), an animal cell, a cell from an invertebrate animal(e.g., fruit fly, cnidarian, echinoderm, nematode, and the like), a cellfrom a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal),a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, arat, a mouse, a non-human primate, a human, and the like). Furthermore,a cell can be a stem cell or a progenitor cell.

As used herein, “stem cell” refers to a cell that has the capacity forself-renewal, i.e., the ability to go through numerous cycles of celldivision while maintaining the undifferentiated state. Stem cells can betotipotent, pluripotent, multipotent, oligopotent, or unipotent. Stemcells can be embryonic, fetal, amniotic, adult, or induced pluripotentstem cells.

As used herein, “induced pluripotent stem cells” refers to a type ofpluripotent stem cell that is artificially derived from anon-pluripotent cell, typically an adult somatic cell, by inducingexpression of specific genes.

“Plant,” as used herein, refers to whole plants, plant organs, planttissues, germplasm, seeds, plant cells, and progeny of the same. Plantcells include, without limitation, cells from seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, roots,shoots, gametophytes, sporophytes, pollen and microspores. Plant partsinclude differentiated and undifferentiated tissues including, but notlimited to roots, stems, shoots, leaves, pollens, seeds, tumor tissueand various forms of cells and culture (e.g., single cells, protoplasts,embryos, and callus tissue). The plant tissue may be in plant or in aplant organ, tissue or cell culture. “Plant organ” refers to planttissue or a group of tissues that constitute a morphologically andfunctionally distinct part of a plant.

“Subject,” as used herein, refers to any member of the phylum Chordata,including, without limitation, humans and other primates, includingnon-human primates such as rhesus macaques, chimpanzees and other monkeyand ape species; farm animals, such as cattle, sheep, pigs, goats andhorses; domestic mammals, such as dogs and cats; laboratory animals,including rabbits, mice, rats and guinea pigs; birds, includingdomestic, wild, and game birds, such as chickens, turkeys and othergallinaceous birds, ducks, and geese; and the like. The term does notdenote a particular age or gender. Thus, adult, young, and newbornindividuals are intended to be covered as well as male and female. Insome embodiments, a host cell is derived from a subject (e.g., stemcells, progenitor cells, tissue specific cells). In some embodiments,the subject is a non-human subject.

The terms “wild-type,” “naturally occurring,” and “unmodified” are usedherein to mean the typical (or most common) form, appearance, phenotype,or strain existing in nature; for example, the typical form of cells,organisms, characteristics, polynucleotides, proteins, macromolecularcomplexes, genes, RNAs, DNAs, or genomes as they occur in, and can beisolated from, a source in nature. The wild-type form, appearance,phenotype, or strain serve as the original parent before an intentionalmodification. Thus, mutant, variant, engineered, recombinant, andmodified forms are not wild-type forms.

As used herein, the terms “engineered,” “genetically engineered,”“recombinant,” “modified,” and “non-naturally occurring” areinterchangeable and indicate intentional human manipulation.

As used herein, “transgenic organism” refers to an organism whose genomeis genetically modified. The term includes the progeny (any generation)of a transgenic organism, provided that the progeny has the geneticmodification.

As used herein, “isolated” can refer to a nucleic acid or polypeptidethat, by the human intervention, exists apart from its nativeenvironment and is therefore not a product of nature. An isolatednucleic acid or polypeptide can exist in a purified form and/or canexist in a non-native environment such as, for example, in a recombinantcell.

In one aspect, the present invention relates to an engineeredcross-type-nucleic-acid targeting nucleic acid (“cross-type-NATNA”). Thecross-type-NATNA typically comprises a first NATNA that targets a firstprotein to site-specifically bind a first nucleic acid target sequenceconnected with a second NATNA that targets a second protein tosite-specifically bind a second nucleic acid target sequence. Theconnection can be covalent or non-covalent. The first protein preferablycomprises one or more catalytically active nuclease domains and/or oneor more catalytically inactive nuclease domains. The second proteinpreferably comprises one or more catalytically active nuclease domainsand/or one or more catalytically inactive nuclease domains. In someembodiments, the cross-type-NATNA comprises a first CRISPR Cas-NATNAfrom a first type of CRISPR Cas system (e.g., a Cas-NATNA that iscapable of forming a complex with a Cas protein from a CRISPR Type I,II, III, IV, V, or VI system) connected with a second type of CRISPRCas-NATNA from a second CRISPR Cas system (e.g., a Cas-NATNA that iscapable of forming a complex with a Cas protein from a CRISPR Type I,II, III, IV, V, or VI system different from the first CRISPR Cassystem).

In other embodiments, the cross-type-NATNA comprises a first CRISPRCas-NATNA from a Class 1 CRISPR Cas system (e.g., a Cas-NATNA that iscapable of forming a complex with a Cas protein from a CRISPR Type I,III, or IV system) connected with a second CRISPR Cas-NATNA from aCRISPR Class 2 Cas system (e.g., a Cas-NATNA that is capable of forminga complex with a Cas protein from a CRISPR Type II, V, or VI system). Inadditional embodiments, the cross-type-NATNA comprises a first CRISPRCas-NATNA from a Class 1 CRISPR Cas system (e.g., a Cas-NATNA that iscapable of forming a complex with a Cas protein from a CRISPR Type I,III, or IV system) connected with a different, second CRISPR Cas-NATNAfrom a CRISPR Class 1 CRISPR Cas system (e.g., a Cas-NATNA that iscapable of forming a complex with a Cas protein from a CRISPR Type I,III, or IV system). In further embodiments, a cross-type-NATNA comprisesan Ago-NATNA and either a CRISPR Class 1 NATNA or a CRISPR Class 2NATNA.

A preferred embodiment of the present invention includes an engineeredCRISPR Class 2 cross-type-nucleic-acid targeting nucleic acid (“CRISPRClass 2 cross-type-NATNA”), comprising a Cpf1-NATNA, a first Cas9-NATNA,connected with a second Cas9-NATNA, wherein the first Cas9-NATNA or thesecond Cas9-NATNA connected covalently or non-covalently with theCpf1-NATNA.

In some embodiments, two different NATNAs are connected covalently toform the cross-type-NATNA. For example, a first Cas9-NATNA or a secondCas9-NATNA is connected covalently with a Cpf1-NATNA, and the Cpf1-NATNAis covalently bound to the 5′ end or the 3′ end of the first Cas9-NATNA,or the 5′ end or the 3′ end of the second Cas9-NATNA. In furtherembodiments, the different NATNAs are covalently joined through a linkerelement nucleotide sequence. For example, a first Cas9-NATNA or a secondCas9-NATNA is connected covalently with a Cpf1-NATNA through a linkerelement nucleotide sequence, and the Cpf1-NATNA is covalently boundthrough the linker element nucleotide sequence to the 5′ end or the 3′end of the first Cas9-NATNA, or the 5′ end or the 3′ end of the secondCas9-NATNA.

FIG. 8, FIG. 9, and FIG. 10 each illustrate an example of suchcovalently attachments using RNA NATNAs.

FIG. 8 illustrates an example of an engineered CRISPR Class 2cross-type-NATNA wherein the 5′ end of a Cpf1-crRNA is covalently linkedto the 3′ end of a Cas9-crRNA. In the figure, the arrow indicates thelinkage region between the Cpf1-crRNA and the Cas9-crRNA. The Cpf1-crRNAand the Cas9-crRNA can be directly linked to each other by a covalentbond or, for example, linked to each other through a linker elementnucleotide sequence (e.g., 5′ end of a Cpf1-crRNA is covalently linkedto the 3′ end of a linker element nucleotide sequence and the 5′ end ofthe linker element nucleotide sequence is covalently linked to the 3′end of a Cas9-crRNA).

FIG. 9 illustrates an example of an engineered CRISPR Class 2cross-type-NATNA wherein the 3′ end of a Cpf1-crRNA is covalently linkedto the 5′ end of a Cas9-tracrRNA. In the figure, the arrow indicates thelinkage region between the Cpf1-crRNA and the Cas9-tracrRNA. TheCpf1-crRNA and the Cas9-tracrRNA can be directly linked to each other bya covalent bond or, for example, linked to each other through a linkerelement nucleotide sequence (e.g., 3′ end of a Cpf1-crRNA is covalentlylinked to the 5′ end of a linker element nucleotide sequence and the 3′end of the linker element nucleotide sequence is covalently linked tothe 5′ end of a Cas9-tracrRNA).

FIG. 10 illustrates an example of an engineered CRISPR Class 2cross-type-NATNA wherein the 3′ end of a Cpf1-crRNA is covalently linkedto the 5′ end of a Cas9-crRNA. In the figure, the arrow indicates thelinkage between the Cpf1-crRNA and the Cas9-crRNA. The Cpf1-crRNA andthe Cas9-crRNA can be directly linked to each other by a covalent bondor, for example, linked to each other through a linker elementnucleotide sequence (e.g., 3′ end of a Cpf1-crRNA is covalently linkedto the 5′ end of a linker element nucleotide sequence and the 3′ end ofthe linker element nucleotide sequence is covalently linked to the 5′end of a Cas9-crRNA).

In some embodiments, the cross-type-NATNA comprises two differentNATNAs, wherein one of the NATNAs comprises more than one polynucleotide(e.g., FIG. 8, FIG. 9, FIG. 10). The NATNA comprising more than onepolynucleotide can be covalently linked through a loop element to form asingle-NATNA (e.g., compare FIG. 1B, a Cas9-crRNA/Cas9-tracrRNA withFIG. 2, a Cas9-sgRNA).

For example, the 3′ end of a first Cas9-NATNA can be connectedcovalently through a loop element with the 5′ end of a second Cas9-NATNAforming a single-Cas9-associated nucleic-acid targeting nucleic acid(“single-Cas9-NATNA”) having a 5′ end and a 3′ end. Thus, thesingle-Cas9-NATNA comprises the first Cas9-NATNA and the secondCas9-NATNA. The single-Cas9-NATNA is covalently attached to a Cpf1-NATNAto form a CRISPR Class 2 cross-type-NATNA. FIG. 4, FIG. 6, and FIG. 7each illustrate an example of such covalently attachments using RNANATNAs.

FIG. 4 illustrates an example of an engineered CRISPR Class 2cross-type-NATNA wherein the 5′ end of a Cpf1-crRNA is covalently linkedto the 3′ end of a Cas9-sgRNA. In the figure, the arrow indicates thelinkage region between the Cpf1-crRNA and the Cas9-sgRNA. The Cpf1-crRNAand the Cas9-sgRNA can be directly linked to each other by a covalentbond or, for example, linked to each other through a linker elementnucleotide sequence (e.g., 5′ end of a Cpf1-crRNA is covalently linkedto the 3′ end of a linker element nucleotide sequence and the 5′ end ofthe linker element nucleotide sequence is covalently linked to the 3′end of a Cas9-sgRNA).

FIG. 6 illustrates an example of an engineered CRISPR Class 2cross-type-NATNA wherein the 5′ end of a Cpf1-crRNA is covalently linkedto the 3′ end of a Cas9-sgRNA, and wherein one of the 3′ hairpinelements (e.g., FIG. 2, 207, 208) is removed (i.e., deleted) from theCas9-sgRNA. In the figure, the arrow indicates the linkage regionbetween the Cpf1-crRNA and the Cas9-sgRNA. The Cpf1-crRNA and theCas9-sgRNA can be directly linked to each other by a covalent bond or,for example, linked to each other through a linker element nucleotidesequence (e.g., 5′ end of a Cpf1-crRNA is covalently linked to the 3′end of a linker element nucleotide sequence and the 5′ end of the linkerelement nucleotide sequence is covalently linked to the 3′ end of aCas9-sgRNA, wherein one of the 3′ hairpins is removed from theCas9-sgRNA).

FIG. 7 illustrates an example of an engineered CRISPR Class 2cross-type-NATNA wherein the 5′ end of a Cpf1-crRNA is covalently linkedto the 3′ end of a Cas9-sgRNA, and wherein both 3′ hairpin elements(e.g., FIG. 2, 207, 208) are removed from the Cas9-sgRNA. In the figure,the arrow indicates the linkage region between the Cpf1-crRNA and theCas9-sgRNA. The Cpf1-crRNA and the Cas9-sgRNA can be directly linked toeach other by a covalent bond or, for example, linked to each otherthrough a linker element nucleotide sequence (e.g., 5′ end of aCpf1-crRNA is covalently linked to the 3′ end of a linker elementnucleotide sequence and the 5′ end of the linker element nucleotidesequence is covalently linked to the 3′ end of a Cas9-sgRNA, wherein thesecond hairpin and third hairpin are removed from the Cas9-sgRNA).

As discussed above, in some embodiments, each polynucleotide of each ofthe two different NATNAs (see, e.g., FIG. 1B, FIG. 2, and FIG. 3)further comprises one or more covalently connected linker elements atthe 5′ end and/or the 3′ end. For example, a Cpf1-NATNA furthercomprises one or more covalently connected linker elements at the 5′ endand/or the 3′ end, a first Cas9-NATNA (e.g., a Cas9-crRNA) and/or asecond Cas9-NATNA (e.g., a Cas9-tracrRNA) further comprises one or morecovalently connected linker elements at the 5′ end and/or the 3′ end ofthe first Cas9-NATNA and/or the second Cas9-NATNA, and asingle-Cas9-NATNA (e.g., a Cas9-sgRNA) further comprises one or morecovalently connected linker elements at the 5′ end and/or the 3′ end.

In other embodiments, two different NATNAs are connected non-covalentlyto form the cross-type-NATNA. For example, an engineered CRISPR Class 2cross-type-NATNA can comprise a first Cas9-NATNA or a second Cas9-NATNAconnected non-covalently with a Cpf1-NATNA through hydrogen base-pairbonding at the 5′ end or 3′ end of the first Cas9-NATNA, or the 5′ endor 3′ end of the second Cas9-NATNA. In additional embodiments, fornon-covalent connection, the different NATNAs are non-covalentlyconnected through one or more linker element sequences.

FIG. 5A and FIG. 5B illustrate examples of such non-covalentlyattachments using RNA NATNAs.

FIG. 5A illustrates an example of an engineered Cas9-Cpf1-NATNA. In thisexample, the 3′ end of a Cas9-sgRNA is modified to remove a 3′ hairpinelement (FIG. 2, 207 or 208). The Cpf1-stem RNA sequence 1 (FIG. 3,303), including the sequence 5′ of the Cpf1 stem (about 3-6nt),important for pseudo-knot formation, is covalently linked to theCas9-sgRNA that was modified to remove a 3′ hairpin. The Cpf1-NATNA ismodified to remove the Cpf1-stem RNA sequence 1 (FIG. 3, 303), loopelement (FIG. 3, 304), and the sequence 5′ of the Cpf1 stem. TheCpf1-stem RNA sequence 1 (FIG. 3, 303) can hybridize through hydrogenbase-pair bonding with the Cpf1-crRNA stem RNA sequence 2 (FIG. 3, 305).In the figure, the arrow indicates the region of hydrogen bondingbetween the Cpf1-stem RNA sequence 2 of the Cpf1-crRNA and theCas9-sgRNA modified to comprise the Cpf1-stem RNA sequence 1, includingthe sequence 5′ of the Cpf1 stem.

In another embodiment, additional sequences can be added to the 3′ endof the Cas9-sgRNA and/or additional complementary or non-complementarysequences can be added to the 5′ end of the Cpf1-crRNA. FIG. 5Billustrates an example of such an engineered Cas9-Cpf1-NATNA. In thisexample, the 3′ end of a Cas9-sgRNA is modified by covalent attachmentof a first linker element sequence and the 5′ end of a Cpf1-crRNA ismodified by covalent attachment of a second linker element sequence,wherein the first linker element and the second linker element hybridizeand form an connection through hydrogen base-pair bonding. In thefigure, the arrow indicates the region of hydrogen bonding between thefirst linker element and the second linker element.

In addition to covalent linkages and non-covalent linkages, other typesof connections to form a cross-type-NATNA can be used between the firstNATNA and the second NATNA including, but not limited to ligand/ligandbinding moiety pairings, and/or cross-linking. Ligand/ligand bindingmoiety pairings can be useful to form a cross-type-NATNA include, butare not limited to: a selected nucleic acid sequence and a correspondingaptamer; and a nucleic acid secondary structure/a small molecule, ion,or protein that binds to the nucleic acid secondary structure.Typically, a first NATNA is adapted to comprise a ligand (e.g., thefirst NATNA comprises at its 3′ end a selected nucleic acid sequence)and a second NATNA is adapted to comprise a ligand binding moiety (e.g.,the second NATNA comprises an aptamer at its 5′ end that binds theselected nucleic acid sequence).

Cross-linking agents useful to form a cross-type-NATNA (by cross-linkinga first NATNA to a second NATNA) include, but are not limited to:alkylating agents (e.g., 1, 3-bis(2-chloroethyl)-1-nitrosourea) andnitrogen mustard); cisplatin (cis-diamminedichloroplatinum(II)) and itsderivatives); ionizing radiation; nitrous acid; reactive chemicals(e.g., malondialdehyde); psoralens (activated in the presence of UV);and aldehydes (e.g., acrolein and crotonaldehyde).

In preferred embodiments, the present invention includes an engineeredcross-type-NATNA (comprising a first NATNA and a second NATNA) whereinthe first NATNA is capable of forming a first complex with a firstprotein and the second NATNA is capable of forming a second complex withthe second protein. Furthermore, if the first complex forms, it iscapable of targeting site-specific binding to a first nucleic acidtarget sequence and, if the second complex forms, it is capable oftargeting site-specific binding to a second nucleic acid targetsequence. For example, an engineered CRISPR Class 2 cross-type-NATNA,comprising a Cpf1-NATNA and a Cas9-NATNA, is capable of forming aCpf1-NATNA/Cpf1 protein complex with a Cpf1 protein and Cas9-NATNA/Cas9protein complex with a Cas9 protein (the entire nucleoprotein complex isreferred to as a Cas9-Cpf1-NATNA/Cas9&Cpf1 protein complex). When theCas9-Cpf1-NATNA/Cpf1 protein complex is formed between theCas9-Cpf1-NATNA and a Cpf1 protein, the complex is capable of binding afirst double-stranded nucleic acid target sequence; and when theCas9-Cpf1-NATNA/Cas9 protein complex is formed between the Cas9-NATNA(e.g., the first Cas9-NATNA and the second Cas9-NATNA or thesingle-Cas9-NATNA) and a Cas9 protein, the Cas9-Cpf1-NATNA/Cas9 proteincomplex is capable of binding a second double-stranded nucleic acidtarget sequence. Thus, when the Cas9-Cpf1-NATNA/Cas9&Cpf1 proteincomplex is formed, the Cas9-Cpf1-NATNA/Cas9&Cpf1 protein complex iscapable of binding the first double-stranded nucleic acid targetsequence and the second double-stranded nucleic acid target sequence.

Example 1 describes production of exemplary components of engineeredcross-type-NATNAs.

Example 5 describes identification and screening of Class 2 crRNAs thatcan be used to make engineered cross-type-NATNAs of the presentinvention. Example 6 describes identification and screening of Class 2tracrRNAs that can be used to make engineered cross-type-NATNAs of thepresent invention.

Example 8 describes a method of probing for sites in Class 2 Type Vguide crRNA backbones that are tolerant of modification. Example 9describes a method of probing for sites tolerant of modification inClass 2 Type II Cas9 guide RNA backbones. The information obtained bythe methods of Example 8 and Example 9 provides guidance regarding sitesin the backbones through which individual NATNAs can be connected toform engineered cross-type-NATNAs of the present invention.

In a second aspect, the present invention is directed to nucleicacid/protein compositions comprising a cross-type-NATNA (comprising afirst NATNA and a second NATNA), a first protein with which the firstNATNA is capable of forming a complex, and a second protein with whichthe second NATNA is capable of forming a complex; thus thecross-type-NATNA, the first protein, and the second protein are capableof forming a cross-type-NATNA/first&second protein complex. Typically,the first protein comprises one or more nuclease activities, and thesecond protein comprises one or more nuclease activities. In someembodiments, the first protein is catalytically inactive for one or moreof the nuclease activities, the second protein is catalytically inactivefor one or more of the nuclease activities, or both the first protein iscatalytically inactive for one or more of the nuclease activities andthe second protein is catalytically inactive for one or more of thenuclease activities. Other embodiments of thecross-type-NATNA/first&second protein complex, wherein either the firstprotein or the second protein is catalytically inactive, includeassociation of a donor polynucleotide with the catalytically inactiveprotein.

In one embodiment of this second aspect of the present invention, anucleic acid/protein composition comprises an engineered CRISPR Class 2cross-type-NATNA as described herein, and a Cas9 protein, a Cpf1protein, or both a Cas9 protein and a Cpf1 protein. In anotherembodiment, the engineered CRISPR Class 2 cross-type-NATNA is in acomplex with the Cas9 protein (Cas9-Cpf1-NATNA/Cas9 protein complex),the Cpf1 protein (Cas9-Cpf1-NATNA/Cpf1 protein complex), or both theCas9 protein and the Cpf1 protein (Cas9-Cpf1-NATNA/Cas9&Cpf1 proteinscomplex). The Cas9 protein and the Cpf1 protein can have combinations ofthe following endonuclease activities: for the Cas9 protein, both theRuvC-1 and HNH domains of the Cas9 protein can be catalyticallyinactive, the RuvC-1 domain of the Cas9 protein can be catalyticallyinactive, the HNH domain of the Cas9 protein can be catalyticallyinactive, and both the RuvC-1 and HNH domains of the Cas9 protein can becatalytically inactive; and for the Cpf1 protein, the Cpf1 protein canbe catalytically active or catalytically inactive.

In some embodiments of the composition, either the Cas9 protein or theCpf1 protein is catalytically inactive (dCas9 or dCpf1 ) and thecomposition further comprises a donor polynucleotide wherein the donorpolynucleotide comprises a nucleotide sequence complementary to thespacer element, or the regions adjacent to the spacer element, of theCpf1-NATNA when dCpf1 is present, or a nucleotide sequence complementaryto the spacer element, or the regions adjacent to the spacer element, ofthe Cas9-NATNA when dCas9 is present. The donor polynucleotide iscapable of associating with the spacer element, or the regions adjacentto the spacer element, through hydrogen bonding between the donorpolynucleotide nucleotide sequence complementary to the spacer element,or the sequence adjacent to the spacer element.

Mutations of the Cas9 protein that are enzymatically inactive forRuvC-1-related nuclease activity, HNH-related nuclease activity, andboth RuvC-1-related nuclease activity and HNH-related nuclease activityare known in the art. Mutations of the Cpf1 protein that areenzymatically inactive are known in the art (see, e.g., Yamano, T., etal., Cell 165(4):949-962 (2016)); Zetsche, B., et al., Cell 163:1-13(2015)). Enzymatically inactive Argonaute proteins can be made bymodification to one or both of the Argonaute nuclease domains (e.g., thePIWI and/or PAZ domains). A nuclease-deficient Argonaute protein can beengineering through sequence modification of the catalytic residues ofnuclease domains, particularly the PIWI domain, of the Argonaute protein(Jinek, M., et al., Nature 457:405-412 (2009)). Additionally, naturallyoccurring nuclease-deficient Argonaute proteins are known in the art(e.g., with inactive PIWI domains), and can be used as models to designinactive variants of nuclease-active Argonautes (see, e.g., Makarova,K., et al., Biology Direct 4:29 (2009) [doi:10.1186/1745-6150-4-29]).

Across CRISPR systems, “guide biogenesis” (also referred to as “guideprocessing”) involves endonuclease or exonuclease truncation of theguide RNA sequence following transcription of the CRISPR array.Enzymatic processing of the guide RNA can be carried out by RNasesencoded by the Cas operon (e.g., Cas6 of Class 1 Type I-E systems) or byendogenous RNases (e.g., RNase III of Class 2 Type II-A systems).

In Class 2 Type V systems, guide biogenesis is performed by the Cpf1protein nuclease. The Cpf1 protein is also responsible forsequence-specific double-stranded DNA target cleavage.

In the Type V system, cleavage of the pre-crRNA (see, e.g., FIG. 14B)occurs in an upstream region (e.g., in a 5′ direction) from thepseudo-knot secondary structure and results in the generation of a guideCpf1 crRNA (see, e.g., FIG. 14A). In some embodiments of the presentinvention, preventing the Cpf1 protein from cleaving 5′ of the guidecrRNA stem element is useful, for example, to prevent separation of aCas9-Cpf1 NATNA/Cas9&Cpf1 protein complex into a Cas9-NATNA/Cas9 proteincomplex and a Cpf1-NATNA/Cpf1 protein complex occurring as a result ofCpf1 protein cleavage of the Cpf1 NATNA/Cas9&Cpf1 protein complex. Ithas been demonstrated that the sequence of Type V pre-crRNA can bemodified to prevent guide RNA processing by the Type V CRISPR Cpf1protein (see Fonfara, I., et al., Nature 532(7600):517-521 (2016)).

One method to prevent Cpf1 cleavage of sequences 5′ of the guide crRNAstem element is by modification (e.g., base mutations, insertions,deletions, or chemical modifications) of the bases in the regionupstream of the pseudo-knot or within the pseudo-knot of the pre-crRNAto prevent the processing of the pre-crRNA by the Cpf1 protein. Toevaluate the effect of such modifications on guide processing, themodified pre-crRNA is incubated in the presence of a cognate Cpf1protein for a period of time in a suitable buffer. The mixture istreated with Proteinase K (Denville Scientific, South Plainfield, NJ) toremove the protein and the mixture is analyzed by polyacrylamide gelelectrophoresis to evaluate whether cleavage of the modified pre-crRNAoccurs. A pre-crRNA not incubated in the presence of a cognate Cpf1protein serves as positive control (i.e., a control for the absence ofguide processing). If no single modification in the pre-crRNA issufficient to ablate guide processing, then combinations ofmodifications exhibiting reduced processing of the pre-crRNA can becombined into a pre-crRNA design and retested for the absence of guideprocessing activity. Modifications of pre-crRNA that result in theinability of the modified pre-crRNA to be processed can be furtherevaluated for the ability of the Cpf1-pre-crRNA/Cpf1 protein complex tomaintain sequence-specific binding and/or cleavage of a DNA targetnucleic acid comprising the pre-crRNA spacer element.

A second method to prevent Cpf1 cleavage of sequences 5′ of the guidecrRNA stem element is by modification of the Cpf1 protein. In thismethod, the amino acid residues of the Cpf1 protein are modified toperturb guide processing. X-ray crystallography of guide crRNA/Cpf1protein complexes has shown that the pseudo-knot is bound by theinterface of two protein domains designated the wedge domain (WED) andthe RuvC domain (see Yamano, T., et al., Cell 165(4):949-962 (2016).Amino acid residues of Cpf1 proximal to the region binding the 5′ end ofthe guide crRNA and/or the pseudo-knot structure are likely to beinvolved in endonuclease catalysis of pre-crRNAs. Mutagenesisstrategies, such as alanine screening (see, e.g., Lefevre, F., et al.,Nucleic Acids Research 25(2):447-448 (1997); Lee, et al., MolecularPharmacology 50(1):140-148 (1996)) can be used to modify regions withinthe WED and RuvC domain, or other domains within the Cpf1 protein, toidentify residues in the protein responsible for guide crRNA processing.In this method, Cpf1 proteins comprising alanine mutations can beexpressed and incubated with a cognate pre-crRNA in a suitable buffer.After incubation, Proteinase K is added to the reaction mix to removethe Cpf1 protein and the reaction mix is then analyzed by polyacrylamidegel electrophoresis to evaluate whether cleavage of the modifiedpre-crRNA occurred. A pre-crRNA not incubated in the presence of acognate Cpf1 protein serves as positive control (i.e., a control for theabsence of guide processing). If no single mutation in the Cpf1 proteinis sufficient to ablate guide processing, then combinations of mutationsexhibiting reduced processing of the pre-crRNA can be combined into asingle Cpf1 protein construct and retested for the absence of guideprocessing activity. Candidate mutations or combinations of mutations inthe Cpf1 protein can be further evaluated for the ability of theCpf1-pre-crRNA complex to maintain sequence-specific binding and/orcleavage of a DNA target nucleic acid comprising the pre-crRNA spacerelement.

In a third aspect, the present invention relates to nucleic acidsequences encoding a cross-type-NATNA (comprising a first NATNA and asecond NATNA), as well as expression cassettes, vectors, and recombinantcells comprising nucleic acid sequences encoding cross-type-NATNAs. Oneembodiment of the third aspect of the present invention relates to oneor more nucleic acid sequences encoding an engineered CRISPR Class 2cross-type-NATNA. In some embodiments of the third aspect of theinvention, such expression cassettes, vectors, and recombinant cellsfurther comprise sequences encoding a first protein (e.g., a Cpf1protein) with which the first NATNA is capable of forming a complex,and/or a second protein (e.g., a Cas9 protein) with which the secondNATNA is capable of forming a complex.

In one embodiment, the present invention relates to one or moreexpression cassettes comprising one or more nucleic acid sequencesencoding an engineered cross-type-NATNA, and optionally one or morenucleic acid sequences encoding a first protein with which thecross-type-NATNA is capable of forming a complex, and/or a secondprotein with which the cross-type-NATNA is capable of forming a complex(e.g., one or more nucleic acid sequences encoding an engineered CRISPRClass 2 cross-type-NATNA, and optionally one or more nucleic acidsequences encoding a Cpf1 and/or a Cas9 protein). Expression cassettestypically comprise regulatory sequences that are involved in one or moreof the following: regulation of transcription, post-transcriptionalregulation, and regulation of translation. Expression cassettes can beintroduced into a wide variety of organisms including bacterial cells,yeast cells, plant cells, and mammalian cells. Expression cassettestypically comprise functional regulatory sequences corresponding to theorganism(s) into which they are being introduced.

In some embodiments, one or more nucleic acid sequences encoding anengineered cross-type-NATNA, and optionally one or more nucleic acidsequences encoding a first protein with which the cross-type-NATNA iscapable of forming a complex, and/or a second protein with which thecross-type-NATNA is capable of forming a complex (e.g., an engineeredCRISPR Class 2 cross-type-NATNA, and optionally one or more nucleic acidsequences encoding a Cpf1 and/or a Cas9 protein) are operably linked toregulatory elements.

A further embodiment of the present invention relates to vectors,including expression vectors, comprising one or more nucleic acidsequences encoding an engineered cross-type-NATNA, and optionally one ormore nucleic acid sequences encoding a first protein with which thecross-type-NATNA is capable of forming a complex, and/or a secondprotein with which the cross-type-NATNA is capable of forming a complex(e.g., one or more nucleic acid sequences encoding an engineered CRISPRClass 2 cross-type-NATNA, and optionally one or more nucleic acidsequences encoding a Cpf1 and/or a Cas9 protein). Vectors can alsoinclude sequences encoding selectable or screenable markers.Furthermore, nuclear targeting sequences can also be added, for example,to Cas9 protein and Cpf1 protein coding sequences. Vectors can alsoinclude polynucleotides encoding protein tags (e.g., poly-His tags,hemagglutinin tags, fluorescent protein tags, bioluminescent tags). Thecoding sequences for such protein tags can be fused to, for example, theone or more nucleic acid sequences encoding a Cas9 protein and/or a Cpf1protein.

General methods for construction of expression vectors are known in theart. Expression vectors for host cells are commercially available. Thereare several commercial software products designed to facilitateselection of appropriate vectors and construction thereof, such asinsect cell vectors for insect cell transformation and gene expressionin insect cells, bacterial plasmids for bacterial transformation andgene expression in bacterial cells, yeast plasmids for celltransformation and gene expression in yeast and other fungi, mammalianvectors for mammalian cell transformation and gene expression inmammalian cells or mammals, and viral vectors (including lentivirus,retrovirus, adenovirus, herpes simplex virus I or II, parvovirus,reticuloendotheliosis virus, and adeno-associated virus (AAV) vectors)for cell transformation and gene expression and methods to easily allowcloning of such polynucleotides. Illustrative plant transformationvectors include those derived from a Ti plasmid of Agrobacteriumtumefaciens (Lee, L. Y., et al., Plant Physiology 146(2): 325-332(2008)). Also useful and known in the art are Agrobacterium rhizogenesplasmids. For example, SNAPGENE™ (GSL Biotech LLC, Chicago, Ill.;snapgene.com/resources/plasmid files/ your time is valuable/) providesan extensive list of vectors, individual vector sequences, and vectormaps, as well as commercial sources for many of the vectors.

Lentiviral vectors are examples of vectors useful for introduction intomammalian cells of one or more nucleic acid sequences encoding anengineered cross-type-NATNA, and optionally one or more nucleic acidsequences encoding a first protein with which the cross-type-NATNA iscapable of forming a complex, and/or a second protein with which thecross-type-NATNA is capable of forming a complex (e.g., one or morenucleic acid sequences encoding an engineered CRISPR Class 2cross-type-NATNA, and optionally one or more nucleic acid sequencesencoding a Cpf1 and/or a Cas9 protein). Lentivirus is a member of theRetroviridae family and is a single-stranded RNA virus, which can infectboth dividing and non-dividing cells as well as provide stableexpression through integration into the genome. To increase the safetyof lentivirus, components necessary to produce a viral vector are splitacross multiple plasmids. Transfer vectors are typically replicationincompetent and may additionally contain a deletion in the 3′LTR, whichrenders the virus self-inactivating after integration. Packaging andenvelope plasmids are typically used in combination with a transfervector. For example, a packaging plasmid can encode combinations of theGag, Pol, Rev, and Tat genes. A transfer plasmid can comprise viral LTRsand the psi packaging signal. The envelope plasmid comprises an envelopeprotein (usually vesicular stomatitis virus glycoprotein, VSV-GP,because of its wide infectivity range).

Lentiviral vectors based on human immunodeficiency virus type-1 (HIV-1)have additional accessory proteins that facilitate integration in theabsence of cell division. HIV-1 vectors have been designed to address anumber of safety concerns. These include separate expression of theviral genes in trans to prevent recombination events leading to thegeneration of replication-competent viruses. Furthermore, thedevelopment of self-inactivating vectors reduces the potential fortransactivation of neighboring genes and allows the incorporation ofregulatory elements to target gene expression to particular cell types(see, e.g., Cooray, S., et al., Methods in Enzymology 507:29-57 (2012)).

Transformed host cells (or recombinant cells) are cells or the progenyof cells that have been transformed or transfected, using recombinantDNA techniques, with one or more nucleic acid sequences encoding anengineered cross-type-NATNA, and optionally one or more nucleic acidsequences encoding a first protein with which the cross-type-NATNA iscapable of forming a complex, and/or a second protein with which thecross-type-NATNA is capable of forming a complex (e.g., one or morenucleic acid sequences encoding an engineered CRISPR Class 2cross-type-NATNA, and optionally one or more nucleic acid sequencesencoding a Cpf1 and/or a Cas9 protein). Methods of introducingpolynucleotides (e.g., an expression vector) into host cells are knownin the art and are typically selected based on the kind of host cell.Such methods include, for example, viral or bacteriophage infection,transfection, conjugation, electroporation, calcium phosphateprecipitation, polyethyleneimine-mediated transfection, DEAE-dextranmediated transfection, protoplast fusion, lipofection, liposome-mediatedtransfection, particle gun technology, direct microinjection, andnanoparticle-mediated delivery.

As an alternative to expressing one or more nucleic acid sequencesencoding an engineered cross-type-NATNA, and optionally one or morenucleic acid sequences encoding a first protein with which thecross-type-NATNA is capable of forming a complex, and/or a secondprotein with which the cross-type-NATNA is capable of forming a complex,a cross-type-NATNA and/or the first and second protein can be directlyintroduced into a cell, for example. Or one or more components can beexpressed by a cell and the other component(s) directly introduced.Methods to introduce the components into a cell include electroporation,lipofection, and ballistic gene transfer (e.g., using a gene gun or abiolistic particle delivery system).

A variety of exemplary host cells are disclosed herein that can be usedto produce recombinant cells by introduction of one or more nucleic acidsequences encoding an engineered cross-type-NATNA, and optionally one ormore nucleic acid sequences encoding a first protein with which thecross-type-NATNA is capable of forming a complex, and/or a secondprotein with which the cross-type-NATNA is capable of forming a complex(e.g., one or more nucleic acid sequences encoding an engineered CRISPRClass 2 cross-type-NATNA, and optionally one or more nucleic acidsequences encoding a Cpf1 and/or a Cas9 protein. Such host cellsinclude, but are not limited to a plant cell, a yeast cell, a bacterialcell, an insect cell, an algal cell, or a mammalian cell.

Methods of introducing polynucleotides (e.g., an expression vector) intohost cells to produce recombinant cells are known in the art and aretypically selected based on the kind of host cell. Such methods include,for example, viral or bacteriophage infection, transfection,conjugation, electroporation, calcium phosphate precipitation,polyethyleneimine-mediated transfection, DEAE-dextran mediatedtransfection, protoplast fusion, lipofection, liposome-mediatedtransfection, particle gun technology, direct microinjection, andnanoparticle-mediated delivery. For ease of discussion, “transfection”is used below to refer to any method of introducing polynucleotides intoa host cell.

Preferred methods for introducing polynucleotides plant cells includemicroprojectile bombardment and Agrobacterium-mediated transformation.Alternatively, other non-Agrobacterium species (e.g., Rhizobium) andother prokaryotic cells that are able to infect plant cells andintroduce heterologous polynucleotides into the genome of the infectedplant cell can be used. Other methods include electroporation,liposome-mediated transfection, transformation using pollen or viruses,and chemicals that increase free DNA uptake, or free DNA delivery usingmicroprojectile bombardment. See, e.g., Narusaka, Y., et al., Chapter 9,in Transgenic Plants—Advances and Limitations, edited by Yelda, O., ISBN978-953-51-0181-9 (2012).

In some embodiments, a host cell is transiently or non-transientlytransfected. In some embodiments, a cell is transfected as it naturallyoccurs in a subject. In some embodiments, a cell that is transfected istaken from a subject, e.g., a primary cell or progenitor cell. In someembodiments, the primary cell or progenitor cell is cultured and/or isreturned after ex vivo transfection to the same subject (autologoustreatment) or to a different subj ect.

The cross-type-NATNA/first&second protein complexes described herein canbe used to generate non-human transgenic organisms by site-specificallyintroducing a selected polynucleotide sequence at a DNA target locus inthe genome to generate a modification of the genomic DNA. The transgenicorganism can be an animal or a plant.

A transgenic animal is typically generated by introducing the systeminto a zygote cell. A basic technique, described with reference tomaking transgenic mice (Cho, A., et al., “Generation of TransgenicMice,” Current Protocols in Cell Biology, CHAPTER.Unit-19.11 (2009)),involves five basic steps: first, preparation of a system, as describedherein, including a suitable donor polynucleotide; second, harvesting ofdonor zygotes; third, microinjection of the system into the mousezygote; fourth, implantation of microinjected zygotes intopseudo-pregnant recipient mice; and fifth, performing genotyping andanalysis of the modification of the genomic DNA established in foundermice. The founder mice will pass the genetic modification to anyprogeny. The founder mice are typically heterozygous for the transgene.Mating between these mice will produce mice that are homozygous for thetransgene 25% of the time.

Methods for generating transgenic plants are also well known. Atransgenic plant generated, e.g., using Agrobacterium transformationmethods, typically contains one transgene inserted into one chromosome.It is possible to produce a transgenic plant that is homozygous withrespect to a transgene by sexually mating (i.e., selfing) an independentsegregant transgenic plant containing a single transgene to itself, forexample an F0 plant, to produce F1 seed. Plants formed by germinating F1seeds can be tested for homozygosity. Typical zygosity assays include,but are not limited to, single nucleotide polymorphism assays andthermal amplification assays that distinguish between homozygotes andheterozygotes.

As an alternative to using a system described herein for the directtransformation of a plant, transgenic plants can be formed by crossing afirst plant that has been transformed with a system with a second plantthat has never been exposed to the system. For example, a first plantline containing a transgene can be crossed with a second plant line tointrogress the transgene into the second plant line, thus forming asecond transgenic plant line.

A fourth aspect of the present invention relates to methods of usingnucleic acid/protein compositions comprising an engineeredcross-type-NATNA, a first protein with which the cross-type-NATNA iscapable of forming a complex, and a second protein with which thecross-type-NATNA is capable of forming a complex. Embodiments of nucleicacid/protein compositions are described herein, for example, in thepreceding second aspect of the invention.

In one embodiment, the present invention includes a method of binding anucleic acid sequence (e.g., DNA), comprising contacting a first nucleicacid target sequence in the nucleic acid (e.g., DNA) and a secondnucleic acid target sequence in the nucleic acid sequence (e.g., DNA)with a nucleic acid/protein composition comprising an engineeredcross-type-NATNA in a complex with a first protein with which thecross-type-NATNA forms a complex, and a second protein with which thecross-type-NATNA forms a complex (e.g., an engineered CRISPR Class 2cross-type-NATNA, a Cpf1 protein with which the CRISPR Class 2cross-type-NATNA forms a complex, and a Cas9 protein with which theCRISPR Class 2 cross-type-NATNA forms a complex), thereby facilitatingbinding of the nucleic acid/protein composition to the first nucleicacid target sequence in the nucleic acid sequence and the second nucleicacid target sequence in the nucleic acid. The engineeredcross-type-NATNA comprises a first-NATNA spacer element (e.g., aCpf1-NATNA spacer element) that is complementary to the first nucleicacid target sequence (e.g., DNA) and a second-NATNA spacer element(e.g., a Cas9-NATNA spacer element) that is complementary to the secondnucleic acid target sequence (e.g., DNA). In some embodiments thenucleic acid target sequence is DNA or genomic DNA. Such methods ofbinding a nucleic acid target sequence are carried out in vitro, in cell(e.g., in cultured cells), ex vivo (e.g., stem cells removed from asubject), and in vivo.

A variety of methods are known in the art to evaluate and/or quantitateprotein-nucleic acid interactions including, but not limited to, thefollowing: immunoprecipitation (ChIP) assays, DNA electrophoreticmobility shift assays (EMSA), DNA pull-down assays, and microplatecapture and detection assays. Commercial kits, materials, and reagentsare available to practice many of these methods from, for example,Thermo Scientific (Wilmington, Del.), Signosis (Santa Clara, Calif.),Bio-Rad (Hercules, Calif.), and Promega (Madison, Wis.)). A commonapproach to detect protein-nucleic acid interactions is EMSA (see, e.g.,Hellman L. M., et al., Nature Protocols 2(8):1849-1861 (2007)).

In another embodiment, the present invention includes a method ofcutting a nucleic acid sequence (e.g., DNA), comprising contacting afirst nucleic acid target sequence in the nucleic acid (e.g., DNA) and asecond nucleic acid target sequence in the nucleic acid sequence (e.g.,DNA) with a nucleic acid/protein composition comprising an engineeredcross-type-NATNA in a complex with a first protein with which thecross-type-NATNA forms a complex, and a second protein with which thecross-type-NATNA forms a complex (e.g., an engineered CRISPR Class 2cross-type-NATNA, a Cpf1 protein with which the CRISPR Class 2cross-type-NATNA forms a complex, and a Cas9 protein with which theCRISPR Class 2 cross-type-NATNA forms a complex), thereby facilitatingbinding of the nucleic acid/protein composition to the first nucleicacid target sequence in the nucleic acid sequence and the second nucleicacid target sequence in the nucleic acid. The engineeredcross-type-NATNA comprises a first-NATNA spacer element (e.g., aCpf1-NATNA spacer element) that is complementary to the first nucleicacid target sequence (e.g., DNA) and a second-NATNA spacer element(e.g., a Cas9-NATNA spacer element) that is complementary to the secondnucleic acid target sequence (e.g., DNA). The first protein (e.g., Cpf1) of the bound nucleic acid/protein composition cuts the first nucleicacid target sequence, and the second protein (e.g., Cas9) of the boundnucleic acid/protein composition cuts the second nucleic acid targetsequence. In some embodiments the nucleic acid target sequence is DNA orgenomic DNA. Such methods of binding a nucleic acid target sequence arecarried out in vitro, in cell (e.g., in cultured cells), ex vivo (e.g.,stem cells removed from a subject), and in vivo.

Example 3 describes performance of Cas protein-mediated cleavage assays.Example 2 describes how to produce double-stranded DNA target regionsfor use in the cleavage assays.

Example 4 presents a deep sequencing analysis for detection of targetmodifications in eukaryotic cells using cross-type-NATNAs of the presentinvention. Example 7 presents an alternative analysis, the T7E1 assay,for detection of target modifications in eukaryotic cells usingcross-type-NATNAs of the present invention.

Example 10 illustrates the use of cross-type-NATNAs of the presentinvention to select and modify DNA target sequences present in genomicDNA and to measure the level of cleavage activity at those sites.

A method of cutting a nucleic acid target sequence is exemplified inFIG. 11A, FIG. 11B, FIG. 11C, and FIG. 11D. These figures present anexample of excision of a sequence from a nucleic acid target using anengineered Cas9-Cpf1-NATNA and Cas9 and Cpf1 proteins both having activeendonucleases. FIG. 11A shows a Cas9 protein (FIG. 11A, 1101) and a Cpf1protein (FIG. 11A, 1102), an engineered Cas9-Cpf1-NATNA as shown in FIG.4 (FIG. 11A, 1103), and a double-stranded nucleic acid (FIG. 11A, 1104)comprising a first DNA target binding sequence complementary to theCas9-sgRNA spacer element (FIG. 11A, 1105) and a second DNA targetbinding sequence complementary to the Cpf1-crRNA spacer element (FIG.11A, 1106). FIG. 11B illustrates the Cas9 (FIG. 11B, 1101) and Cpf1(FIG. 11B, 1102) proteins in complex with the engineered Cas9-Cpf1-NATNA(FIG. 11B, 1103) and the hydrogen bonding of a first DNA target bindingsequence to the Cas9-sgRNA spacer element (FIG. 11B, 1107) and a secondDNA target binding sequence to the Cpf1-crRNA spacer element (FIG. 11B,1109). In the figure, the double-stranded nucleic acid is indicated asFIG. 11B, 1104, the Cas9 PAM sequence in the double-stranded nucleicacid is indicated as FIG. 11B, 1108 and the Cpf1 PAM sequence isindicated as FIG. 11B, 1110. FIG. 11C illustrates double-strandblunt-end cuts made by Cas9 at the first DNA target binding sequence(FIG. 11C, 1111) and the double-strand staggered cuts made by Cpf1 atthe second DNA target binding sequence (FIG. 11C, 1112). Thedouble-stranded nucleic acid is indicated as FIG. 11C, 1104. FIG. 11Dillustrates an excised nucleic acid sequence (FIG. 11D, 1113) that isstill associated with the engineered Cas9-Cpf1-NATNA complex (FIG.11D,1114). One end of the nucleic acid target comprises a staggereddouble-strand break resulting from Cpf1 cleavage (FIG. 11D, 1112) andthe other an essentially blunt end double-strand break resulting fromCas9 cleavage (FIG. 11D, 1111). The double-stranded nucleic acid isindicated as FIG. 11D, 1104. In other embodiments, the Cas9 and Cpf1proteins are deficient in endonuclease activity and the binding of theengineered Cas9-Cpf1-NATNA complex to a nucleic acid target can be used,for example, to block transcription and subsequent expression of a geneadjacent to the nucleic acid target.

In yet another embodiment, the present invention includes a method ofmodifying DNA in a cell, comprising contacting a first DNA targetsequence in the DNA and a second DNA target sequence in the DNA with anucleic acid/protein composition comprising an engineeredcross-type-NATNA in a complex with a first protein with which thecross-type-NATNA forms a complex, and a second protein with which thecross-type-NATNA forms a complex (e.g., an engineered CRISPR Class 2cross-type-NATNA, a Cpf1 protein with which the CRISPR Class 2cross-type-NATNA forms a complex, and a Cas9 protein with which theCRISPR Class 2 cross-type-NATNA forms a complex), thereby facilitatingbinding of the nucleic acid/protein composition to the first nucleicacid target sequence in the nucleic acid sequence and the second nucleicacid target sequence in the nucleic acid. The engineeredcross-type-NATNA comprises a first-NATNA spacer element (e.g., aCpf1-NATNA spacer element) that is complementary to the first DNA targetsequence and a second-NATNA spacer element (e.g., a Cas9-NATNA spacerelement) that is complementary to the second DNA target sequence (e.g.,DNA). The first protein (e.g., Cpf1 ) of the bound nucleic acid/proteincomposition cuts the first DNA target sequence, and the second protein(e.g., Cas9) of the bound nucleic acid/protein composition cuts thesecond DNA target sequence. The cell repairs the first cut site and thesecond cut site. Cell DNA repair pathways include, HDR, NHEJ, MMEJ. Insome embodiments the nucleic acid target sequence is DNA or genomic DNA.Such methods of binding a nucleic acid target sequence are carried outin vitro, in cell (e.g., in cultured cells), ex vivo (e.g., stem cellsremoved from a subject), and in vivo. The contracting step may furthercomprise a donor polynucleotide being present, wherein at least aportion of the donor polynucleotide is incorporated between the firstcut site and the second cut site.

In another embodiment, the invention relates to a method to bring adonor polynucleotide into proximity of a double-strand break in anucleic acid target, typically DNA, in a cell. The method comprisescontacting a first DNA target sequence in the DNA and a second DNAtarget sequence in a donor polynucleotide with a nucleic acid/proteincomposition comprising an engineered cross-type-NATNA in a complex witha first protein with which the cross-type-NATNA forms a complex, and asecond protein with which the cross-type-NATNA forms a complex (e.g., anengineered CRISPR Class 2 cross-type-NATNA, a Cpf1 protein with whichthe CRISPR Class 2 cross-type-NATNA forms a complex, and a Cas9 proteinwith which the CRISPR Class 2 cross-type-NATNA forms a complex), therebyfacilitating binding of the nucleic acid/protein composition to thefirst DNA target sequence in the DNA and the second DNA target sequencein the donor polynucleotide. The first DNA target sequence iscomplementary to a first NATNA spacer (e.g., a Cpf1-NATNA spacer elementor a Cas9-NATNA spacer element), wherein the associated first protein isa catalytically active nuclease protein (e.g., a Cpf1 protein or a Cas9protein, respectively). The second DNA target sequence is complementaryto a second NATNA spacer (e.g., a Cpf1-NATNA spacer element or aCas9-NATNA spacer element), wherein the associated second protein is acatalytically inactive nuclease protein (e.g., a dCpf1 protein or adCas9 protein, respectively). For example, when the catalytically activenuclease protein is a Cpf1 protein, the catalytically inactive nucleaseprotein is a dCas9 protein, and vice versa. The catalytically activenuclease protein of the bound nucleic acid/protein composition cuts thefirst DNA target sequence to form a cut site. The donor polynucleotideis in proximity to the cut site (e.g., the double-strand break) becausethe catalytically active nuclease protein and the catalytically inactivenuclease protein are complexed with the cross-type-NATNA, that is, theyare part of the same nucleic acid/protein composition. In someembodiments, at least a portion of the donor polynucleotide isintroduced into the cut site in the DNA (e.g., by an HDR repair process)resulting in modifying the DNA.

FIG. 12A, FIG. 12B, and FIG. 12C illustrate using an engineeredCas9-Cpf1-NATNA and Cas9 and Cpf1 proteins, wherein endonuclease domainsof Cas9 are active and the endonuclease domain of Cpf1 is inactive, tobring a donor polynucleotide into proximity of a double-strand break ina nucleic acid target. FIG. 12A shows a Cas9 protein (FIG. 12A, 1201)and a Cpf1 protein (FIG. 12A, 1202), an engineered Cas9-Cpf1-NATNA asshown in FIG. 4 (FIG. 12A, 1203), and a double-stranded nucleic acid(FIG. 12A, 1204) comprising a first DNA target binding sequencecomplementary to the Cas9-sgRNA spacer element (FIG. 12A, 1205) and adonor polynucleotide (FIG. 12A, 1207) comprising a second DNA targetbinding sequence complementary to the Cpf1-crRNA spacer element (FIG.12A, 1206). FIG. 12B illustrates the Cas9 (FIG. 12B, 1201) and Cpf1(FIG. 12B, 1202) proteins in complex with the engineered Cas9-Cpf1-NATNA(FIG. 12B, 1203) and the hydrogen bonding of the first DNA targetbinding sequence to the Cas9-sgRNA spacer element (FIG. 12B, 1208) andthe second DNA target binding sequence in the donor polynucleotide tothe Cpf1-crRNA spacer element (FIG. 12B, 1210). In the figure, the Cas9PAM sequence in the double-stranded nucleic acid is indicated as FIG.12B, 1209 and the Cpf1 PAM sequence in the donor polynucleotide isindicated as FIG. 12B, 1211. The double-stranded nucleic acid isindicated as FIG. 12B, 1204, and the donor polynucleotide is indicatedas FIG. 12B, 1207. FIG. 12C illustrates double-strand blunt-end cutsmade by Cas9 at the first DNA target binding sequence (FIG. 12C, 1212)and shows the donor polynucleotide (FIG. 12C, 1207) in proximity to thedouble-strand blunt-end cuts. Having the donor polynucleotide (FIG. 12C,1207) in close proximity to the double-strand cuts (FIG. 12C, 1212)increases the likelihood of integration of the donor polynucleotidesequences, or portions thereof, into the double-stranded nucleic acidcomprising the nucleic acid target (FIG. 12C, 1204). In someembodiments, the endonuclease domain of Cpf1 is active and capable ofbinding to and cleaving the donor molecule, but Cpf1 does notdisassociate from the donor after cleavage.

FIG. 13A, FIG. 13B, and FIG. 13C illustrate using an engineeredCas9-Cpf1-NATNA and Cas9 and Cpf1 proteins, wherein endonuclease domainsof Cas9 are inactive and the endonuclease domain of Cpf1 is active, tobring a donor polynucleotide into proximity of a double-strand break ina nucleic acid target. FIG. 13A shows a Cas9 protein (FIG. 13A, 1301)and a Cpf1 protein (FIG. 13A, 1302), an engineered Cas9-Cpf1-NATNA asshown in FIG. 4 (FIG. 13A, 1303), and a double-stranded nucleic acid(FIG. 13A, 1304) comprising a first DNA target binding sequencecomplementary to the Cpf1-crRNA spacer element (FIG. 13A, 1306) and adonor polynucleotide (FIG. 13A, 1307) comprising a second DNA targetbinding sequence complementary to the Cas9-sgRNA spacer element (FIG.13A, 1305). FIG. 13B illustrates the Cas9 (FIG. 13B, 1301) and Cpf1(FIG. 13B, 1302) proteins in complex with the engineered Cas9-Cpf1-NATNA(FIG. 13B, 1303) and the hydrogen bonding of the first DNA targetbinding sequence to the Cpf1-crRNA spacer element (FIG. 13B, 1308) andthe second DNA target binding sequence in the donor polynucleotide tothe Cas9-sgRNA spacer element (FIG. 13B, 1310). In FIG. 13B, the Cpf1PAM sequence in the double-stranded nucleic acid is indicated as 1309and the Cas9 PAM sequence in the donor polynucleotide is indicated as1311. The double-stranded nucleic acid comprising the first DNA targetbinding sequence is indicated as FIG. 13B, 1304. FIG. 13C illustratesdouble-strand staggered cuts made by Cpf1 at the first DNA targetbinding sequence (FIG. 13C, 1312) and shows the donor polynucleotide(FIG.13C, 1307) in proximity to the double-strand staggered cuts. Havingthe donor polynucleotide (FIG. 13C, 1307) in close proximity to thedouble-strand cuts (FIG. 13C, 1312) increases the likelihood ofintegration of the donor polynucleotide sequences, or portions thereof,into the double-stranded nucleic acid comprising the first DNA targetbinding sequence (FIG. 13C, 1304). In some embodiments, the endonucleasedomains of Cas9 are active and capable of binding to and cleaving thedonor molecule, but Cas9 does not disassociate from the donor aftercleavage.

In a further embodiment, the invention relates to a method of bringing afirst nucleic acid target site, typically DNA, into the proximity of asecond nucleic acid target site, typically DNA, in a cell. The methodcomprises contacting a first nucleic target sequence and a secondnucleic target sequence with a nucleic acid/protein compositioncomprising an engineered cross-type-NATNA in a complex with a firstprotein with which the cross-type-NATNA forms a complex, and a secondprotein with which the cross-type-NATNA forms a complex (e.g., anengineered CRISPR Class 2 cross-type-NATNA, a Cpf1 protein with whichthe CRISPR Class 2 cross-type-NATNA forms a complex, and a Cas9 proteinwith which the CRISPR Class 2 cross-type-NATNA forms a complex), therebyfacilitating binding of the nucleic acid/protein composition to thefirst nucleic acid target sequence and the second nucleic acid targetsequence. The first DNA target sequence is complementary to a firstNATNA spacer (e.g., a Cpf1-NATNA spacer element or a Cas9-NATNA spacerelement), wherein the associated first protein is a catalyticallyinactive nuclease protein (e.g., a dCpf1 protein or a dCas9 protein,respectively). The second DNA target sequence is complementary to asecond NATNA spacer (e.g., a Cpf1-NATNA spacer element or a Cas9-NATNAspacer element), wherein the associated second protein is acatalytically inactive nuclease protein (e.g., a dCpf1 protein or adCas9 protein, respectively). For example, if the first protein is adCpf1 protein, the second protein is a dCas9 protein, and vice versa.The first nucleic acid target site is brought into proximity of a secondnucleic acid target site because the first and second catalyticallyinactive nuclease proteins are complexed with the cross-type-NATNA, thatis, they are part of the same nucleic acid/protein composition. In someembodiments, the first nucleic acid target sequence and the secondnucleic acid target sequence are on separate polynucleotides (e.g.,different chromosomes) or a single polynucleotide comprises the firstnucleic acid target sequence and the second nucleic acid target sequence(e.g., different sections of the same chromosome).

In yet another embodiment, the present invention also includes methodsof modulating in vitro or in vivo transcription, for example,transcription of a gene comprising regulatory element sequences. Themethod comprises contacting a first nucleic target sequence and a secondnucleic target sequence with a nucleic acid/protein compositioncomprising an engineered cross-type-NATNA in a complex with a firstprotein with which the cross-type-NATNA forms a complex, and a secondprotein with which the cross-type-NATNA forms a complex (e.g., anengineered CRISPR Class 2 cross-type-NATNA, a Cpf1 protein with whichthe CRISPR Class 2 cross-type-NATNA forms a complex, and a Cas9 proteinwith which the CRISPR Class 2 cross-type-NATNA forms a complex), therebyfacilitating binding of the nucleic acid/protein composition to thefirst nucleic acid target sequence and the second nucleic acid targetsequence. At least one of the first DNA target sequence and the secondDNA target sequence comprise the regulatory element sequences. The firstDNA target sequence is complementary to a first NATNA spacer (e.g., aCpf1-NATNA spacer element or a Cas9-NATNA spacer element), wherein theassociated first protein is a catalytically inactive nuclease protein(e.g., a dCpf1 protein or a dCas9 protein, respectively). The second DNAtarget sequence is complementary to a second NATNA spacer (e.g., aCpf1-NATNA spacer element or a Cas9-NATNA spacer element), wherein theassociated second protein is a catalytically inactive nuclease protein(e.g., a dCpf1 protein or a dCas9 protein, respectively). For example,if the first protein is a dCpf1 protein, the second protein is a dCas9protein, and vice versa. In addition, the first and/or second proteincan be fusion proteins, for example, dCas9 fused to a repressor oractivator domain, and/or dCpf1 fused to a repressor or activator domain.The binding of the nucleic acid/protein composition to the first DNAtarget sequence and the second DNA target sequence modulatestranscription of the gene. In some embodiments, the first DNA targetsequence and the second DNA target sequence comprise the regulatoryelement sequences, and the first DNA target sequence comprises apromoter and the second DNA target sequence comprises a transcriptionstart site.

Any of the components of the nucleic acid/protein compositionscomprising an engineered cross-type-NATNA of the present invention ornucleic acid sequences encoding such components, as described above, canbe incorporated into a kit, optionally including one or more reagents.In some embodiments, a kit includes a package with one or morecontainers holding the kit elements, as one or more separatecompositions or, optionally, as admixture wherein the compatibility ofthe components will allow. In some embodiments, kits also comprise abuffer, a buffering agent, a salt, a sterile aqueous solution, and/orpreservatives. Illustrative kits comprise an engineered CRISPR Class 2cross-type-NATNA, and optionally a Cpf1 and/or a Cas9 protein, and oneor more nucleic acid sequences encoding an engineered CRISPR Class 2cross-type-NATNA, and optionally one or more nucleic acid sequencesencoding a Cpf1 and/or a Cas9 protein.

Furthermore, kits can further comprise instructions for using componentsof the nucleic acid/protein compositions comprising an engineeredcross-type-NATNA of the present invention or nucleic acid sequencesencoding such components. Instructions included in kits of the inventioncan be affixed to packaging material or can be included as a packageinsert. Although the instructions are typically written or printedmaterials, they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to,electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), RF tags, and the like.Instructions can also include the address of an internet site thatprovides the instructions.

Another aspect of the invention relates to methods of making ormanufacturing an engineered cross-type-NATNA or a nucleic acid/proteincomposition comprising an engineered cross-type-NATNA of the presentinvention. In one embodiment, the methods of making or manufacturingcomprises chemically synthesizing an engineered cross-type-NATNA. Insome embodiments, an engineered cross-type-NATNA comprises RNA bases andcan be generated from DNA templates using in vitro transcription.

A nucleic acid/protein composition comprising an engineeredcross-type-NATNA (e.g., an engineered CRISPR Class 2 cross-type-NATNA)can further comprise a detectable label, including a moiety that canprovide a detectable signal. Examples of detectable labels include, butare not limited to, an enzyme, a radioisotope, a member of a specificbinding pair, a fluorophore (FAM), a fluorescent protein (greenfluorescent protein, red fluorescent protein, mCherry, tdTomato), an DNAor RNA aptamer together with a suitable fluorophore (enhanced GFP(EGFP), “Spinach”), a quantum dot, an antibody, and the like. A largenumber and variety of suitable detectable labels are well-known to oneof ordinary skill in the art.

A nucleic acid/protein composition comprising an engineeredcross-type-NATNA (e.g., an engineered CRISPR Class 2 cross-type-NATNA)or cells modified by use of a nucleic acid/protein compositioncomprising an engineered cross-type-NATNA, as described herein, can beused as a pharmaceutical composition formulated, for example, with apharmaceutically acceptable excipient. Illustrative excipients includecarriers, stabilizers, diluents, dispersing agents, suspending agents,thickening agents, and the like. The pharmaceutical composition canfacilitate administration of a nucleic acid/protein compositioncomprising an engineered cross-type-NATNA to an organism. Pharmaceuticalcompositions can be administered in therapeutically effective amounts byvarious forms and routes including, for example, intravenous,subcutaneous, intramuscular, oral, aerosol, parenteral, ophthalmic, andpulmonary administration.

Numerous advantages are obtained using an engineered cross-type-NATNA ofthe present invention, comprising a first-NATNA and a second-NATNA,wherein a first protein is capable of forming a complex with thefirst-NATNA of the cross-type-NATNA, and a second protein is capable offorming a complex with the second-NATNA of the cross-type-NATNA (e.g.,an engineered CRISPR Class 2 cross-type-NATNA, comprising a Cpf1-NATNAand a Cas9-NATNA, wherein a Cpf1 protein is capable of forming a complexwith the Cpf1-NATNA of the CRISPR Class 2 cross-type-NATNA, and a Cas9protein is capable of forming a complex with the Cas9-NATNA of theCRISPR Class 2 cross-type-NATNA) including, but not limited to:

-   -   a reduction in off-targeting binding using a nucleic        acid/protein composition comprising an engineered        cross-type-NATNA (e.g., a Cas9-Cpf1-NATNAs), relative to a        first-NATNA/first protein complex alone (e.g., a Cpf1-NATNA/Cpf1        protein complex) or a second-NATNA/second protein complex alone        (e.g., a Cas9-NATNA/Cas9 protein complex);    -   tethering of a donor polynucleotide through use of a nucleic        acid/protein composition comprising an engineered        cross-type-NATNA (e.g., a Cas9-Cpf1-NATNA) to bring the donor        polynucleotide into proximity of a cut in a double-stranded        nucleic acid;    -   bringing two separate polynucleotides (e.g., two different        chromosomes) or two regions of a single polynucleotide (e.g.,        two regions of a single chromosome) into proximity of each other        using a nucleic acid/protein composition comprising an        engineered cross-type-NATNA (e.g., a Cas9-Cpf1-NATNA); and    -   transcriptional modulation of a target gene by binding of a        nucleic acid/protein composition comprising an engineered        cross-type-NATNA (e.g., a Cas9-Cpf1-NATNA) to regulatory        sequences operably linked to the target gene.

Yet another advantage of a nucleic acid/protein composition comprisingan engineered cross-type-NATNA (e.g., a Cas9-Cpf1-NATNA) is eliminatingthe need of a PAM sequence for cleavage of a nucleic acid target. Forexample, a first nucleic acid target sequence complementary to a firstspacer element (see, e.g., FIG. 13A, 1305) is selected. The firstnucleic acid target sequence comprises a PAM sequence for the firstprotein (e.g., a Cpf1 PAM). A second nucleic acid target sequencecomplementary to a second spacer element (see, e.g., FIG. 13A, 1306) isselected, wherein the second nucleic acid target sequence lacks a PAMsequence for the second protein (e.g., a Cas9 PAM) and is in closeproximity to the first nucleic acid targeting sequence. The firstprotein (e.g., a Cpf1 protein) in nucleic acid/protein compositioncomprising an engineered cross-type-NATNA (e.g., a Cas9-Cpf1-NATNA) isendonuclease inactive (e.g., a dCpf1 protein) and the second protein hasan active endonuclease (e.g., a Cas9 protein). The local unwindingcaused by the inactive endonuclease protein binding to the first nucleicacid target can allow cleavage at the second nucleic acid target site bythe active endonuclease protein even in the absence of a PAM for theactive endonuclease protein. (Similarly an endonuclease inactive Cas9and an endonuclease active Cpf1 can be used.)

Various embodiments contemplated herein include, but are not be limitedto, one or more of the following. The embodiments are numbered for easeof reference.

Embodiment 1. An engineered three-element nucleic-acid targeting nucleicacid (NATNA), comprising: a Cpf1-associated NATNA comprising a spacerelement, a 5′ end, and a 3′ end; a Cas9-associated first NATNAcomprising a spacer element, a 5′ end, and a 3′ end; and aCas9-associated second NATNA comprising a tracr element, a 5′ end, and a3′ end; wherein the Cas9-associated first NATNA and the Cas9-associatedsecond NATNA are associated through hydrogen base-pair bonding; andwherein Cpf1-associated NATNA is connected with the 5′ end or the 3′ endof the Cas9-associated first NATNA comprising a spacer element, or the5′ end or the 3′ end of the Cas9-associated second NATNA comprising atracr element.

Embodiment 2. The engineered three-element NATNA of embodiment 1,wherein a complex formed between the Cpf1-associated NATNA and a Cpf1protein is capable of binding a first double-stranded nucleic acidtarget sequence; and wherein a complex formed between theCas9-associated first NATNA, the Cas9-associated second NATNA, and aCas9 protein is capable of binding a second double-stranded nucleic acidtarget sequence.

Embodiment 3. The engineered three-element NATNA of embodiment 1 or 2,wherein the Cpf1-associated NATNA is connected with the 5′ end or 3′ endof the Cas9-associated first NATNA, or the 5′ end or 3′ end of theCas9-associated second NATNA, and the connection is through hydrogenbonding.

Embodiment 4. The engineered three-element NATNA of embodiment 3,wherein the Cpf1-associated NATNA further comprises a covalentlyconnected linker element at the 5′ end and/or the 3′ end of theCpf1-associated NATNA.

Embodiment 5. The engineered three-element NATNA of embodiment 3 or 4,wherein the Cas9-associated first NATNA and/or the Cas9-associatedsecond NATNA further comprises a covalently connected linker element atthe 5′ end and/or the 3′ end of the Cas9-associated first NATNA and/orthe Cas9-associated second NATNA.

Embodiment 6. The engineered three-element NATNA of embodiment 1 or 2,wherein the Cpf1-associated NATNA is connected with the 5′ end or the 3′end of the Cas9-associated first NATNA, or the 5′ end or the 3′ end ofthe Cas9-associated second NATNA, and the connection is through acovalent bond.

Embodiment 7. The engineered three-element NATNA of embodiment 6,wherein the Cpf1-associated NATNA further comprises a covalentlyconnected linker element at the 5′ end and/or the 3′ end of theCpf1-associated NATNA.

Embodiment 8. The engineered three-element NATNA of embodiment 6 or 7,wherein the Cas9-associated first NATNA and/or the Cas9-associatedsecond NATNA further comprises a covalently connected linker element atthe 5′ end and/or the 3′ end of the Cas9-associated first NATNA and/orthe Cas9-associated second NATNA.

Embodiment 9. The engineered three-element NATNA of embodiment 1 or 2,wherein the 3′ end of the Cas9-associated first NATNA is covalentlyconnected through a loop element with the 5′ end of the Cas9-associatedsecond NATNA forming a single-Cas9-associated NATNA having a 5′ end anda 3′ end; and wherein the Cpf1-associated NATNA is connected with the 5′end or 3′ end of the single-Cas9-associated NATNA, and the connection isthrough hydrogen bonding.

Embodiment 10. The engineered three-element NATNA of embodiment 9,wherein the Cpf1-associated NATNA further comprises a covalentlyconnected linker element at the 5′ end and/or the 3′ end of theCpf1-associated NATNA.

Embodiment 11. The engineered three-element NATNA of embodiment 9 or 10,wherein the single-Cas9-associated NATNA further comprises a covalentlyconnected linker element at the 5′ end and/or the 3′ end of thesingle-Cas9-associated NATNA.

Embodiment 12. The engineered three-element NATNA of embodiment 1 or 2,wherein the 3′ end of the Cas9-associated first NATNA is covalentlyconnected through a loop element with the 5′ end of the Cas9-associatedsecond NATNA forming a single-Cas9-associated NATNA; and wherein theCpf1-associated NATNA is connected with the 5′ end or 3′ end of thesingle-Cas9-associated nucleic-acid targeting nucleic, and theconnection is through a covalent bond.

Embodiment 13. The engineered three-element NATNA of embodiment 12,wherein the Cpf1-associated NATNA further comprises a covalentlyconnected linker element at the 5′ end and/or of the 3′ end theCpf1-associated NATNA.

Embodiment 14. The engineered three-element NATNA of embodiment 12 or13, wherein the single-Cas9-associated NATNA further comprises acovalently connected linker element at the 5′ end and/or the 3′ end ofthe single-Cas9-associated NATNA.

Embodiment 15. The engineered three-element NATNA of any precedingembodiment, wherein at least one of the following elements comprises aRNA: a Cpf1-associated NATNA; a Cas9-associated first NATNA; or aCas9-associated second NATNA.

Embodiment 16. The engineered three-element NATNA of any precedingembodiment, wherein at least one of the following elements comprises aDNA: a Cpf1-associated NATNA; a Cas9-associated first NATNA; or aCas9-associated second NATNA.

Embodiment 17. A nucleic acid/protein composition comprising:

the engineered three-element NATNA of any preceding embodiment, a Cas9protein, and a Cpf1 protein.

Embodiment 18. The nucleic acid/protein composition of embodiment 17,wherein the engineered three-element NATNA forms a complex with the Cas9protein and the Cpf1 protein.

Embodiment 19. The nucleic acid/protein composition of embodiment 18,wherein the Cpf1 protein is inactive for endonuclease activity.

Docket No.: CBI019.19

Embodiment 20. The nucleic acid/protein composition of embodiment 19,further comprising a donor polynucleotide wherein the donorpolynucleotide comprises a nucleotide sequence complementary to theCpf1-associated NATNA spacer element.

Embodiment 21. The nucleic acid/protein composition of embodiment 20,wherein the Cpf1-associated NATNA spacer element is connected with thedonor polynucleotide by hydrogen bonding between the donorpolynucleotide nucleotide sequence complementary to the spacer elementand the spacer element.

Embodiment 22. The nucleic acid/protein composition of embodiment 18,wherein the Cas9 protein is inactive for endonuclease activities.

Embodiment 23. The nucleic acid/protein composition of embodiment 22,further comprising a donor polynucleotide wherein the donorpolynucleotide comprises a nucleotide sequence complementary to theCas9-associated first NATNA spacer element.

Embodiment 24. The nucleic acid/protein composition of embodiment 23,wherein the Cas9-associated first NATNA spacer element is connected withthe donor polynucleotide by hydrogen bonding between the donorpolynucleotide nucleotide sequence complementary to the spacer elementand the spacer element.

Embodiment 25. The nucleic acid/protein composition of embodiment 18,wherein the Cas9 protein is inactive for endonuclease activities and theCpf1 protein is inactive for endonuclease activity.

Embodiment 26. One or more vectors comprising: nucleotide sequencesencoding the engineered three-element NATNA of any of embodiments 1 to16.

Embodiment 27. A recombinant cell comprising: the one or more vectors ofembodiment 26.

Embodiment 28. A recombinant cell comprising: the engineeredthree-element NATNA of any of embodiments 1 to 16.

Embodiment 29. A recombinant cell comprising: the nucleic acid/proteincomposition of any one of embodiments 17 to 25.

Embodiment 30. The recombinant cell of any of embodiments 27 to 29,wherein the cell is a plant cell, a yeast cell, a bacterial cell, aninsect cell, an algal cell, or a mammalian cell.

Embodiment 31. A method of modifying DNA comprising:

contacting a first DNA target sequence in the DNA and a second DNAtarget sequence in the DNA with the nucleic acid/protein composition ofembodiment 18,

wherein the first DNA target sequence is complementary to theCas9-associated first NATNA spacer element, the second DNA targetsequence is complementary to the Cpf1-associated NATNA spacer element,the nucleic acid/protein composition binds to the first DNA targetsequence and the second DNA target sequence, and the Cas9 protein andthe Cpf1 protein cut the first DNA target sequence and the second DNAtarget sequence, respectively, resulting in a modification of the DNA.

Embodiment 32. A method of modifying DNA comprising:

contacting a first DNA target sequence in the DNA and a second DNAtarget sequence in a donor polynucleotide with the nucleic acid/proteincomposition of embodiment 21,

wherein the first DNA target sequence is complementary to theCas9-associated first NATNA spacer element, the second DNA targetsequence is complementary to the Cpf1-associated NATNA spacer element,the nucleic acid/protein composition binds to the first DNA targetsequence, the Cas9 protein cuts the first DNA target sequence, and atleast a portion of the donor polynucleotide is introduced into the DNAresulting in a modification of the DNA.

Embodiment 33. A method of modifying DNA comprising:

contacting a first DNA target sequence in a donor polynucleotide and asecond DNA target sequence in the DNA with the nucleic acid/proteincomposition of embodiment 24,

wherein the first DNA target sequence is complementary to theCas9-associated first NATNA spacer element, the second DNA targetsequence is complementary to the Cpf1-associated NATNA spacer element,the nucleic acid/protein composition binds to the second DNA targetsequence, the Cpf1 protein cuts the second DNA target sequence, and atleast a portion of the donor polynucleotide is introduced into the DNAresulting in a modification of the DNA.

Embodiment 34. A method of bringing a first DNA target site into theproximity of a second DNA target site comprising:

contacting a first DNA target sequence and a second DNA target sequencewith the nucleic acid/protein composition of embodiment 25,

wherein the first DNA target sequence is complementary to theCas9-associated first NATNA spacer element, the second DNA targetsequence is complementary to the Cpf1-associated NATNA spacer element,the nucleic acid/protein composition binds to the first DNA targetsequence and the second DNA target sequence bringing the first DNAtarget site into the proximity of the second DNA target sequence.

Embodiment 35. The method of embodiment 34, wherein the first DNA targetsequence and the second DNA target sequence are on separatepolynucleotides.

Embodiment 36. A method of modulating transcription of a gene comprisingregulatory element sequences, comprising:

contacting a first DNA target sequence and a second DNA target sequencewith the nucleic acid/protein composition of embodiment 25,

wherein the first DNA target sequence is complementary to theCas9-associated first NATNA spacer element, the second DNA targetsequence is complementary to the Cpf1-associated NATNA spacer element,at least one of the first DNA target sequence or the second DNA targetsequence comprise the regulatory element sequences, the nucleicacid/protein composition binds to the first DNA target sequence and thesecond DNA target sequence, and the binding of the nucleic acid/proteincomposition modulates transcription of the gene.

Although preferred embodiments of the present invention have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. From theabove description and the following Examples, one skilled in the art canascertain essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make changes,substitutions, variations, and modifications of the invention to adaptit to various usages and conditions. Such changes, substitutions,variations, and modifications are also intended to fall within the scopeof the present disclosure.

Experimental

Aspects of the present invention are illustrated in the followingExamples. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, concentrations, percent changes, and thelike) but some experimental errors and deviations should be accountedfor. Unless indicated otherwise, temperature is in degrees Centigradeand pressure is at or near atmospheric. It should be understood thatthese Examples are given by way of illustration only and are notintended to limit the scope of what the inventors regard as variousaspects of the present invention.

Materials and Methods

Sequences of oligonucleotide sequences) were provided to commercialmanufacturers for synthesis.

Engineered cross-type-nucleic-acid targeting nucleic acids(“cross-type-NATNAs”; e.g., engineered Cas9-Cpf1 nucleic-acid targetingnucleic acids (“Cas9-Cpf1-NATNAs”)) were assembled by PCR using 3′overlapping primers containing DNA sequences corresponding to across-type-NATNA.

EXAMPLE 1 Production of Components of Cross-Type-Nucleic-Acid TargetingNucleic Acids

This Example describes production of cross-type-NATNAs, for example, aCas9-Cpf1-NATNA similar to the Cas9-Cpf1-NATNA illustrated in FIG. 4.The Cas9-Cpf1-NATNA was designed with a spacer that targeted the codingsequences for vascular endothelial growth factor A (VEGFA) incorporatedupstream (e.g., in a 5′ direction) to the Cas9-sgRNA backbone componentand a spacer that targeted the coding sequences for an indoleamine2,3-dioxygenase 2 (IDO2) incorporated downstream (e.g., in a 3′direction) to the Cpf1 guide RNA backbone component. The Cas9-Cpf1-NATNAwas designed such that the 3′ end of the Cas9 guide RNA sequence waslinked (through a covalent bond) to the 5′ end of the Cpf1 guide RNAsequence to create a single polynucleotide with a continuouspolynucleotide backbone (e.g., FIG. 4).

RNA components were produced by in vitro transcription (e.g., T7 QuickHigh Yield RNA Synthesis Kit, New England Biolabs, Ipswich, MA) from adouble-stranded DNA template incorporating a T7 promoter at the 5′ endof the DNA sequences.

The double-stranded DNA template for the specific Cas9-Cpf1-NATNA(“Cas9-Cpf1-NATNAi”) component used in the following Examples wasassembled by PCR using 3′ overlapping primers containing thecorresponding DNA sequences to the Cas9-Cpf1-NATNA₁ component. Theoligonucleotides used in the assembly are presented in Table 1.

TABLE 1 Overlapping Primers for Generation of Cas9-Cpf1-NATNA₁-Encoding Template SEQ ID NO. Sequence SEQ ID NO. 1AGTGTAATACGACTCACTATAG SEQ ID NO. 2 TAATACGACTCACTATAGGGGTGGGGGGAGTTTGCTCCGTTTTAGAGCTAGAAATAGCAAGTTGAGATAAGG CTAGTCCGTTATCAACTTG SEQ ID NO. 3CCAGGCACTGCTTCTTTCTCTACCATCTACAACAGTAGAAATTAAGCACCGACTCGGTGCCACTTTTTCAAG TTGATAACGGACTAGCCTT SEQ ID NO. 4CCAGGCACTGCTTCTTTCTCTACC

The DNA primers were present at a concentration of 2nM each. Two outerDNA primers corresponding to the T7 promoter (SEQ ID NO. 1) and the 3′end of the RNA sequence (SEQ ID NO. 4) were used at 640nM to drive theamplification reaction. PCR reactions were performed using Q5 Hot StartHigh-Fidelity 2X Master Mix (New England Biolabs, Ipswich, Mass.)following the manufacturer's instructions. PCR assembly reactions werecarried out using the following thermal cycling conditions: 98° C. for 2minutes, 35 cycles of 15 seconds at 98° C., 15 seconds at 60° C., 15seconds at 72° C., and a final extension at 72° C. for 2 minutes. DNAquality was evaluated by agarose gel electrophoresis (1.5%, SYBR® Safe;Life Technologies, Grand Island, N.Y.).

Between 0.25-0.5m of the DNA template for the Cas9-Cpf1-NATNA₁ componentwas transcribed using T7 High Yield RNA Synthesis Kit (New EnglandBiolabs, Ipswich, Mass.) for approximately 16 hours at 37° C.Transcription reactions were treated with DNase I (New England Biolabs,Ipswich, Mass.) and purified using GeneJet RNA Cleanup and ConcentrationKit (Life Technologies, Grand Island, N.Y.). RNA yield was quantifiedusing a NanodropTm 2000 System (Thermo Scientific, Wilmington, Del.).The quality of the transcribed RNA was checked by agarose gelelectrophoresis (2%, SYBR® Safe; Life Technologies, Grand Island, N.Y.).The Cas9-Cpf1-NATNA₁ sequence is shown in Table 2.

TABLE 2 Cas9-Cpf1-NATNA₁ Sequence SEQ ID NO. Sequence* SEQ ID NO.GGGUGGGGGGAGUUUGCUCCGUUUUAGAGCUAGAAAUA 5GCAAGUUGAGAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUAAUUUCUACUGUUGUA GAUGGUAGAGAAAGAAGCAGUGCCUGG*Spacers are underlined

This method for production of Cas9-Cpf1-NATNA₁ can be applied to theproduction of other cross-type-NATNAs described herein.

EXAMPLE 2 Production of Double-Stranded DNA Target Sequences for Use inCleavage Assays

Double-stranded DNA target sequences for use in in vitro Cas cleavageassays were produced using PCR amplification of selected nucleic acidtarget sequences from human genomic DNA.

Double-stranded DNA target sequences (e.g., VEGFA and IDO2) forbiochemical assays were amplified by PCR from phenol-chloroform preparedhuman cell line K562 (American Type Culture Collection (ATCC), Manassas,Va.) genomic DNA (gDNA). PCR reactions were carried out with Q5 HotStart High-Fidelity 2× Master Mix (New England Biolabs, Ipswich, Mass.)following the manufacturer's instructions. 20 ng/pL gDNA in a finalvolume of 25 μl were used to amplify the selected nucleic acid targetsequence under the following conditions: 98° C. for 2 minutes, 35 cyclesof 20 seconds at 98° C., 20 seconds at 60° C., 20 seconds at 72° C., anda final extension at 72° C. for 2 minutes. PCR products were purifiedusing Spin Smart™ PCR purification tubes (Denville Scientific, SouthPlainfield, N.J.) and quantified using a Nanodrop™ 2000 UV-Visspectrophotometer (Thermo Scientific, Wilmington, Del.).

The forward and reverse primers used for amplification of selectedtargeted sequences from gDNA are presented in Table 3.

TABLE 3 Double-Stranded DNA Target Sequence Primer Sequences SEQ ID NO.Sequence SEQ ID NO. 6 CACTCTTTCCCTACACGACGCTCTTCCGATCTCCAGATGGCACATTGTCAGA SEQ ID NO. 7 GGAGTTCAGACGTGTGCTCTTCCGATCTCCTAGTGACTGCCGTCTGC SEQ ID NO. 8 CACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGAACCTGGAGACCATCA SEQ ID NO. 9 GGAGTTCAGACGTGTGCTCTTCCGATCTGAAAGGCACTGAGTGGGAAG

The VEGFA DNA target sequences were amplified using SEQ ID NO. 6 and SEQID NO. 7, yielding a 261bp double-stranded DNA target sequence. IDO2 DNAtarget sequences were amplified using SEQ ID NO. 8 and SEQ ID NO. 9,yielding a 232bp double-stranded DNA target sequence.

Other suitable double-stranded DNA target sequences can be obtainedusing essentially the same method. For non-human nucleic acid targetsequences, genomic DNA from the selected organism (e.g., plant,bacteria, yeast, algae) can be used instead of DNA derived from humancells. Furthermore, polynucleotide sources other than genomic DNA can beused (e.g., vectors and gel isolated DNA fragments).

EXAMPLE 3 Cas Cleavage Assays

This Example illustrates the use of Cas protein/cross-type-NATNAs incleavage assays.

In this Example, a Cas9-Cpf1-NATNA₁/Cas9 protein complex and aCas9-Cpf1-NATNA₁/Cpf1 protein complex were used in in vitro Cas9 or Cpf1cleavage assays, respectively, to evaluate and compare the percentcleavage of selected Cas9-Cpf1-NATNA₁/Cas9 protein complexes andCas9-Cpf1-NATNA₁/Cpf1 protein complexes relative to selecteddouble-stranded DNA target sequences.

The cleavage of double-stranded DNA target sequences was determinedusing the Cas9-Cpf1-NATNA₁ components of Example 1, with one or morecognate Cas proteins to form a complex, against a double-stranded DNAtarget sequence (VEGFA and IDO2; Example 2).

S. pyogenes Cas9 and F. novicida Cpf1 were recombinantly expressed in E.coli and purified for use in an in vitro biochemical cleavage assay.

The Cas9-Cpf1-NATNAi components were diluted to a suitable workingconcentration and incubated for 2 minutes at 95° C., removed from athermocycler and allowed to equilibrate to room temperature.

Cas9-Cpf1-NATNAi was added to either a Cas9 reaction mix or a Cpf1reaction mix. The Cas9 reaction mix comprised Cas9 protein diluted to afinal concentration of 20 nM in reaction buffer (20 mM HEPES, 100 mMKCl, 5 mM MgCl₂, 1 mM DTT, and 5% glycerol at pH 7.4). The Cpf1 reactionmix comprised Cpf1 protein diluted to a final concentration of 50 nM inreaction buffer (20 mM HEPES, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT, and 5%glycerol at pH 7.4). In the reaction mix, the final concentration ofCas9-Cpf1-NATNA₁ was as follows: 200 nM in the Cas9 reaction mix and 500nM in the Cpf1 reaction mix. Each reaction mix was incubated at 37° C.for 10 minutes. The cleavage reaction was initiated by the addition ofthe DNA target sequence to a final concentration of 10 nM. Samples weremixed and centrifuged briefly before being incubated for 15 minutes at37° C. Cleavage reactions were terminated by the addition of ProteinaseK (Denville Scientific, South Plainfield, N.J.) at a final concentrationof 0.2μg/μL and 0.44 mg/μL RNase A Solution (SigmaAldrich, St. Louis,Mo.). Samples were then incubated for 25 minutes at 37° C. and 25minutes at 55° C. 12 μL of the total reaction were evaluated forcleavage activity by agarose gel electrophoresis (2%, SYBR® Gold; LifeTechnologies, Grand Island, N.Y.). For the Cas9 cleavage of a VEGFAdouble-stranded DNA target sequence, the appearance of DNA bands atapproximately 158 bp and approximately 103 bp indicated that cleavage ofthe DNA target sequence had occurred. For the Cpf1 cleavage of an IDO2double-stranded DNA target sequence, the appearance of DNA bands atapproximately 120 bp and approximately 112 bp indicated that cleavage ofthe DNA target sequence had occurred. Cleavage percentages werecalculated using area under the curve (AUC) values as calculated by FIJI(ImageJ; an open source Java image processing program) for each cleavagefragment and the DNA target sequence, and dividing the sum of thecleavage fragments by the sum of both the cleavage fragments and the DNAtarget sequences.

Table 4 presents the results of the Cas9 and Cpf1 cleavage assays usingnucleic acid target sequences VEGFA and IDO2 double-stranded DNA targetsequences.

TABLE 4 Biochemical Cleavage of DNA Target Sequence with aCas9-Cpf1-NATNA Cas9 VEGFA DNA target Cpf1 IDO2 DNA target sequencecleavage sequence cleavage Cas9-Cpf1- 93% 68% NATNA₁

The data presented in Table 4 demonstrate that the Cas9-Cpf1-NATNAi ofthe present invention facilitated Cas protein mediated site-specificcleavage of double-stranded DNA target sequences. The data also showedthat a single-Cas9-Cpf1-NATNA can be used with either a Cas9-nuclease ora Cpf1-nuclease to facilitate the targeted cleavage of a double-strandedDNA target sequence.

Following the guidance of the present specification and Examples, thebiochemical cleavage assay described in this Example can be practiced byone of ordinary skill in the art with other cross-type-NATNAs and theircognate Cas proteins (e.g., Type II CRISPR Cas9 proteins or Type VCRISPR Cpf1 proteins).

EXAMPLE 4 Deep Sequencing Analysis for Detection of Target SequenceModifications in Eukaryotic Cells

This Example illustrates the use of deep sequencing analysis to evaluateand compare the percent cleavage in cells of cross-type-NATNA/Casprotein complexes relative to selected double-stranded DNA targetsequences.

A. Design of a Cas9-Cpf1-NATNA

The human epidermal receptor growth factor 2 (ERBB2) locus was scannedfor 20 nucleotide Cas9 protospacer sequences occurring upstream of a5′-NGG PAM sequence and a 24 nucleotide Cpf1 protospacer sequenceoccurring downstream of a 3′-TTTN PAM sequence. Examples of protospacersequences selected by this method are shown in Table 5.

TABLE 5 ERBB2 Cas9 and Cpf1 Protospacer Sequences Hg38 SEQ IDchromosomal Name NO. Sequence* coordinates Cas9 SEQ IDGTGCCCTCGGTCACACTGTGTG chr17: ERBB2 NO. 10 G 39727008- proto- 39727030spacer Cpf1 SEQ ID TTTCTCTGATGTTCCCTCAACT chr17: ERBB2 NO. 11 GTCACC39727072- proto- 39727099 spacer *Nuclease specific PAMs are underlined

The Cas9 spacer sequence (i.e., the protospacer sequence without thePAM) was incorporated upstream 5′ to a Cas9 sgRNA backbone component andthe Cpf1 spacer sequence (i.e., the protospacer sequence without thePAM) was incorporated downstream 3′ to a Cpf1 guide RNA backbonecomponent to form the cross-type-NATNA ERBB2-Cas9-Cpf1-NATNA. The RNAsequence for this ERBB2-Cas9-Cpf1-NATNA is shown in Table 6.

TABLE 6 ERBB2-Cas9-Cpf1-NATNA Sequence SEQ ID NO. Sequence* SEQ IDGUGCCCUCGGUCACACUGUGGUUUUAGAGCUAGAAAUAGCAA NO. 12GUUGAGAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUAAUUUCUACUGUUGUAGAUUCUGAUGUU CCCUCAACUGUCACC*Nuclease-specific spacers are underlined

Following the guidance of the present specification and Examples,additional cross-type-NATNAs can be designed by one of ordinary skill inthe art.

B. Formation of ERBB2-Cas9-Cpf1-NATNA/Cas9&Cpf1 Protein NucleoproteinComplexes

Primers to assemble the ERBB2-Cas9-Cpf1-NATNA can be ordered from acommercial manufacturer, and the ERBB2-Cas9-Cpf1-NATNA can be assembledessentially as described in Example 1. The RNA sequence for an exemplaryERBB2-Cas9-Cpf1-NATNA is shown in Table 6. S. pyogenes Cas9 andAcidaminococcus sp. Cpf1 are both C-terminally tagged with two nuclearlocalization sequences (NLS) and are recombinantly expressed in E. coli.Ribonucleoprotein (RNP) complexes are formed at a concentration of40pmo1 Cas9 protein:40pmo1 Cpf1 protein:120pmols ERBB2-Cas9-Cpf1-NATNA.Prior to assembly with the Cas9 and Cpf1 proteins, theERBB2-Cas9-Cpf1-NATNA is diluted to the desired concentration (120 pmol)in a final volume of 2 μL, incubated for 2 minutes at 95° C., removedfrom a thermocycler, and allowed to equilibrate to room temperature.Cas9 and Cpf1 proteins are diluted to an appropriate concentration inbinding buffer (20 mM HEPES, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT, and 5%glycerol at pH 7.4) to a final volume of 3pL and are mixed with the 2 μLof ERBB2-Cas9-Cpf1-NATNA followed by incubation at 37° C. for 30minutes.

C. Cell Transfections Using ERBB2-Cas9-Cpf1-NATNA/Cas9&Cpf1 RNP

RNP complexes are transfected into HEK293 cells (ATCC, Manassas Va.),using the Nucleofector® 96-well Shuttle System (Lonza, Allendale, N.J.)and the following protocol. RNP complexes are dispensed in a 5 μpL finalvolume into individual wells of a 96-well plate. The cell culture mediumis removed from the HEK293 cell culture plate and the cells are detachedwith TrypLE™ (Thermo Fisher Scientific, Waltham, Mass.). SuspendedHEK293 cells are pelleted by centrifugation for 3 minutes at 200×g,TrypLE reagents is aspirated, and cells are washed with calcium andmagnesium-free phosphate buffered saline (PBS). Cells are pelleted bycentrifugation for 3 minutes at 200 x g, the PBS is aspirated, and thecell pellet is re-suspended in 10mL of calcium and magnesium-free PBS.

The cells can be counted using the Countess® II Automated Cell Counter(Life Technologies; Grand Island, N.Y.). 2.2×10⁷ cells are transferredto a 1.5 mL microfuge tube and pelleted. The PBS is aspirated and thecells are re-suspended in Nucleofector™ SF (Lonza, Allendale, N.J.)solution to a density of 1×10⁷ cells/mL. 20 μL of the cell suspensionare then added to each individual well containing 5 μL of RNP complexes,and the entire volume from each well is transferred to a well of a96-well Nucleocuvette™ Plate (Lonza, Allendale, N.J.). The plate isloaded onto the Nucleofector™ 96-well Shuttle™ (Lonza, Allendale, N.J.)and cells are nucleofected using the 96-CM-130 Nucleofector™ program(Lonza, Allendale, N.J.). Post-nucleofection, 70 μL Dulbecco's ModifiedEagle Medium (DMEM; Thermo Fisher Scientific, Wilmington, Del.),supplemented with 10% Fetal Bovine Serum (FBS; Thermo Fisher Scientific,Wilmington, Del.), penicillin and streptomycin (Life Technologies, GrandIsland, N.Y.), are added to each well and then 50 μL of the cellsuspension are transferred to a 96-well cell culture plate containing150 μL pre-warmed DMEM complete culture medium. The plate is thentransferred to a tissue culture incubator and maintained at 37° C. in 5%CO₂ for 48 hours.

D. Double-stranded DNA Target Sequence Generation for Deep Sequencing

gDNA is isolated from the HEK293 cells 48 hours after transfection usingthe RNP complexes and 50pL QuickExtract DNA Extraction solution(Epicentre, Madison, Wis.) per well, followed by incubation at 37° C.for 10 minutes, 65° C. for 6 minutes and 95° C. for 3 minutes to stopthe reaction. The isolated gDNA is then diluted with 50 μL sterile waterand samples are stored at −80° C.

Using the isolated gDNA, a first PCR is performed using Q5 Hot StartHigh-Fidelity 2× Master Mix (New England Biolabs, Ipswich, Mass.) at 1×concentration, primers at 0.5 μM each (SEQ ID NO. 13 and SEQ ID NO. 14),3.75 μL of gDNA in a final volume of 10 μL and amplified 98° C. for 1minute, 35 cycles of 10 s at 98° C., 20 seconds at 60° C., 30 seconds at72° C., and a final extension at 72° C. for 2 minutes. Primers aredesigned to amplify the region of the genome targeted by theERBB2-Cas9-Cpf1-NATNA. The PCR reaction is diluted 1:100 in water.

Barcoding PCR is performed using a reaction mix comprising Q5 Hot StartHigh-Fidelity 2× Master Mix (New England Biolabs, Ipswich, Mass.) at 1×concentration; primers at 0.5 μM each (SEQ ID NO. 15 and SEQ ID NO. 16);1 μL of 1:100 diluted first PCR; in a final volume of 10 μL; and thereaction mix is amplified 98° C. for 1 minute, followed 12 cycles of 10s at 98° C., 20 seconds at 60° C., and 30 seconds at 72° C., with afinal extension reaction performed at 72° C. for 2 minutes.

E. SPRIselect Clean-up

The PCR reaction is transferred into a single microfuge tube forSPRIselect (Beckman Coulter, Pasadena, Calif.) bead-based cleanup ofamplicons for sequencing.

To the amplicon, 0.9× volumes of SPRIselect beads are added, mixed, andincubated at room temperature (RT) for 10 minutes. The microfuge tube isplaced on magnetic tube stand (Beckman Coulter, Pasadena, Calif.) untilthe solution clears. Supernatant is removed and discarded, and theresidual beads are washed with 1 volume of 85% ethanol, and incubated atRT for 30 seconds. After incubation, ethanol is aspirated and beads areair dried at RT for 10 minutes. The microfuge tube is then removed fromthe magnetic stand and 0.25× volumes of Qiagen EB buffer (Qiagen, Venlo,Netherlands) is added to the beads, mixed vigorously, and incubated for2 minutes at room temperature. The microfuge tube is returned to themagnet, incubated until the solution had cleared, and supernatantcontaining the purified amplicons is dispensed into a clean microfugetube. The purified amplicon is quantified using a Nanodrop™ 2000 System(Thermo Scientific, Wilmington Del.) and library quality is analyzedusing the Fragment Analyzer™ System (Advanced Analytical Technologies,Ames, Iowa) and the DNF-910 Double-stranded DNA Reagent Kit (AdvancedAnalytical Technologies, Ames, Iowa).

F. Deep Sequencing Set-up

The amplicon is normalized to a 4 nM concentration as calculated fromNanodrop values and size of the amplicons. The library is analyzed onMiSeq Sequencer (Illumina, San Diego, Calif.) with MiSeq Reagent Kit v2(Illumina, San Diego, Calif.) for 300 cycles with two 151-cyclepaired-end runs plus two eight-cycle index reads.

G. Deep Sequencing Data Analysis

The identity of products in the sequencing data is determined based onthe index barcode sequences adapted onto the amplicons in the barcodinground of PCR. A computational script is used to process the MiSeq datathat executes, for example, the following tasks:

-   -   Reads are aligned to the human genome (build GRCh38/38) using        Bowtie (bowtie-bio.sourceforge.net/index.shtml) software.    -   Aligned reads are compared to the expected wild-type ERBB2 locus        sequence, and reads not aligning to any part of the ERBB2 locus        are discarded.    -   Reads matching wild-type ERBB2 sequence are tallied.    -   Reads with indels (insertion or deletion of bases) are        categorized by indel type and tallied.    -   Total indel reads are divided by the sum of wild-type reads and        indel reads to give percent-mutated reads.

Through the identification of indel sequences at the regions targeted bythe ERBB2-Cas9-Cpf1-NATNA/Cas9&Cpf1 protein RNP complexes,sequence-specific targeting in a human cell line can be determined. Therelative distance between the two nucleic acid target sequence sites inthe ERBB2 locus can result in excision of the sequence between the Cas9and Cpf1 nucleic acid target sequences.

Following the guidance of the present specification and Examples, the incell editing of a genomic sequence can be practiced by one of ordinaryskill in the art with other Cas proteins and their cognatecross-type-NATNAs.

EXAMPLE 5

Identification and Screening of crRNAs

This Example describes a method to identify Class 2 crRNAs in differentbacterial species.

This Example describes a method by which crRNAs of species having aClass 2 CRISPR system are identified. The method presented here isadapted from Chylinski, K., et al., RNA Biology 10(5):726-37 (2013). Notall of the following steps are required for screening nor must the orderof the steps be as presented.

A. Identify a Species Containing a Class 2 CRISPR Locus

Using the Basic Local Alignment Search Tool (BLAST,blast.ncbi.nlm.nih.gov/Blast.cgi), a search of the genomes of variousspecies is conducted to identify Class 2 CRISPR Cas nucleases, (e.g.,Cas9 protein, Cpf1 protein, Cas9-like proteins, Cpf1-like proteins,etc.). Class 2 CRISPR systems exhibit a high diversity in sequenceacross species, however Class 2 CRISPR nuclease orthologs have conserveddomains, for example, an HNH endonuclease domain and/or a RuvC/RNase Hdomain. Primary BLAST results are filtered for identified domains,incomplete or truncated sequences are discarded, and species havingClass 2 CRISPR nuclease orthologs are identified.

If a Class 2 CRISPR nuclease ortholog is identified in a species,sequences adjacent to the Cas protein ortholog-coding sequence (e.g.,Cas9 protein or Cpf1 protein) are probed for other Cas proteins and aCas-associated repeat-spacer array is used to identify all sequencesbelonging to the CRISPR-Cas locus. This may be done by alignment toother known Class 2 CRISPR loci.

Once the sequence of the Class 2 CRISPR locus for the nuclease orthologis identified for the species, in silico predictive screening is used toextract the crRNA sequence. The crRNA sequence is contained withinCRISPR repeat array and can be identified by its hallmark repeatingsequences interspaced by foreign spacer sequences.

B. Preparation of RNA-Seq Library

The putative CRISPR array containing the individual crRNA identified insilico is further validated using RNA sequencing (RNA-seq).

Cells from species identified as comprising putative crRNA are procuredfrom a commercial repository (e.g., ATCC, Manassas, Va.; GermanCollection of Microorganisms and Cell Cultures GmbH (DSMZ),Braunschweig, Germany).

Cells are grown to mid-log phase and total RNA prepped using Trizolreagent (SigmaAldrich, St. Louis, Mo.) and treated with DNasel(Fermentas, Vilnius, Lithuania).

10 μg of the total RNA is treated with Ribo-Zero rRNA Removal Kit(Illumina, San Diego, Calif.) and the remaining RNA purified using RNAClean and Concentrators (Zymo Research, Irvine, Calif.).

A library is then prepared using a TruSeq Small RNA Library PreparationKit (Illumina, San Diego, Calif.) following the manufacturer'sinstructions. This results in cDNAs having adapter sequences.

The resulting cDNA library is sequenced using MiSeq Sequencer (Illumina,San Diego, Calif.).

C. Processing of Sequencing Data

Sequencing reads of the cDNA library are processed, for example, usingthe following method.

Adapter sequences are removed using cutadapt 1.1(pypi.python.org/pypi/cutadapt/1.1) and about 15 nt are trimmed from the3′ end of the read to improve read quality.

Reads are aligned to the genome of the respective species (i.e., fromwhich the putative crRNA was identified) with a mismatch allowance of 2nucleotides.

Read coverage is calculated using BedTools(bedtools.readthedocs.org/en/latest/).

Integrative Genomics Viewer (IGV; www.broadinstitute.org/igv/) is usedto map the starting (5′) and ending (3′) position of reads. Total readsretrieved for the putative crRNA are calculated from the SAM file ofalignments.

The RNA-seq data is used to validate that a putative crRNA element isactively transcribed in vivo. Confirmed hits from comparison of the insilico and RNA-seq screens are validated for functional ability tosupport Class 2 CRISPR nuclease cleavage of a double-stranded DNA targetnucleic acid sequences using the methods outline herein (e.g., Examples1, 2, and 3). It is known in the art that the Type V system onlyrequires a crRNA to facilitate Cpf1 nuclease cleavage of adouble-stranded DNA target sequence, whereas the Type II system requiresa crRNA and a cognate tracrRNA to facilitate Cas9 nuclease cleavage of adouble-stranded DNA target sequence. The cognate tracrRNA can beidentified following the method described in Example 6.

Following the guidance of the present specification and Examples, theidentification of novel crRNA sequences related to Cas proteins can bepracticed by one of ordinary skill in the art.

EXAMPLE 6

Identification and Screening of tracrRNA

This Example illustrates a method by which tracrRNAs of species having,for example, a Class 2 Type II CRISPR-Cas9 system can be identified.This is adapted from Chylinski, K., et al., RNA Biology 10(5):726-737(2013). Not all of the following steps are required for screening normust the order of the steps be as presented.

A. Identify a Species Containing a CRISPR-Cas9 Type II System

Using the Basic Local Alignment Search Tool (BLAST,blast.ncbi.nlm.nih.gov/Blast.cgi), a search of the genomes of variousspecies is conducted to identify a Cas9 protein. Class 2 Type IICRISPR-Cas9 systems exhibit a high diversity in sequence across species,however Cas9 orthologs exhibit conserved domain architectures of acentral HNH endonuclease domain and a split RuvC/RNase domain. PrimaryBLAST results are filtered for identified domains; incomplete ortruncated sequences are discarded and Cas9 orthologs are identified.

If a Cas9 ortholog is identified in a species, sequences adjacent to theCas9 ortholog-coding sequence are probed for other Cas proteins and aCas-associated repeat-spacer array to identify all sequences belongingto the CRISPR-Cas9 locus. This may be done by alignment to other knownClass 2 Type II CRISPR-Cas9 loci, with the knowledge that closelyrelated species exhibit similar CRISPR-Cas9 locus architecture (e.g.,Cas protein composition, size, orientation, location of array, locationof tracrRNA, and so on). The tracrRNA element is typically containedwithin the Class 2 Type II CRISPR-Cas9 locus and is readily identifiedby its sequence complementarity to the repeat elements in therepeat-spacer array. The tracr sequences complementary to the repeatelements are called the tracr anti-repeat sequences.

Once the sequence of the CRISPR-Cas9 locus corresponding to the Cas9ortholog is identified for a species, in silico predictive screening isused to extract the tracr anti-repeat sequence to identify theCas-associated tracrRNA. Putative anti-repeats are screened, forexample, as follows.

If the repeat sequence is from a known species, it is identified in andretrieved from the CRISPRdb database (crispr.u-psud.fr/crispr/). If therepeat sequence is not known to be related to a species, repeatsequences are predicted employing CRISPRfinder software(crispr.u-psud.fr/Server/) using the Class 2 Type II CRISPR-Cas9 locusfor the species as described above.

The identified repeat sequence for the species is used to probe theCRISPR-Cas9 locus for the anti-repeat sequence (e.g., using the BLASTpalgorithm or the like). The search is typically restricted to intergenicregions of the CRISPR-Cas9 locus.

An identified tracr anti-repeat region is validated for complementarityto the identified repeat sequence.

A putative anti-repeat region is probed in the regions 5′ and 3′ of theputative anti-repeat region for the presence of a Rho-independenttranscriptional terminator (TransTerm HP, transterm.cbcb.umd.edu/).

By combining the identified sequence comprising the anti-repeat elementand the Rho-independent transcriptional terminator the sequence isdetermined to be the putative tracrRNA of the given species.

B. Preparation of RNA-Seq Library

The in silico identified, putative tracrRNA is further validated usingRNA sequencing (RNA-seq).

Cells from species comprising the putative tracrRNA are procured from acommercial repository (e.g., ATCC, Manassas Va.); DSMZ, Braunschweig,Germany).

Cells are grown to mid-log phase and total RNA prepared using Trizolreagent (SigmaAldrich, St. Louis, Mo.) and treated with DNasel(Fermentas, Vilnius, Lithuania). 10 μg of the total RNA is treated usinga Ribo-Zero rRNA Removal Kit (Illumina, San Diego, Calif.) and theremaining RNA purified using RNA Clean and Concentrators (Zymo Research,Irvine, Calif.).

A library is then prepared using a TruSeq Small RNA Library PreparationKit (Illumina, San Diego, Calif.) following the manufacturer'sinstructions. This results in cDNAs having adapter sequences.

The resulting cDNA library is sequenced using a MiSeq Sequencer(Illumina, San Diego, Calif.).

C. Processing of Sequencing Data

Sequencing reads of the cDNA library are processed, for example, usingthe following method.

Adapter sequences are removed using cutadapt 1.1(pypi.python.org/pypi/cutadapt/1.1) and about 15 nt are trimmed from the3 ′ end of the read to improve read quality.

Reads are aligned to the genome of the respective species (i.e., fromwhich the putative tracrRNA was identified) with a mismatch allowance of2 nucleotides.

Read coverage is calculated using BedTools(bedtools.readthedocs.org/en/latest/).

Integrative Genomics Viewer (IGV, www.broadinstitute.org/igv/) is usedto map the starting (5′) and ending (3′) position of reads. Total readsretrieved for the putative tracrRNA are calculated from the SAM file ofalignments.

The RNA-seq data is used to validate that a putative tracrRNA element isactively transcribed in vivo. Confirmed hits from the comparison of thein silico and RNA-seq screens are validated for functional ability ofthe identified tracrRNA sequence and its cognate crRNA to supportCas9-mediated cleavage of a double-stranded DNA target sequence usingmethods outline herein (e.g., Examples 1, 2, and 3).

Following the guidance of the present specification and Examples, theidentification of novel tracrRNA sequences related to Cas9 proteins canbe accomplished by one of ordinary skill in the art.

EXAMPLE 7

T7E1 Assay for Detection of Target Sequence Modifications in EukaryoticCells

This Example illustrates the use of T7E1 assays to evaluate and comparethe percent cleavage in cell of Cas9-Cpf1-NATNA/Cas9&Cpf1 complexesrelative to selected double-stranded DNA target sequences.

A. Cell Transfections Using Cas Polynucleotide Components

The Cas9-Cpf1-NATNAs are transfected into HEK293 cells constitutivelyexpressing S. pyogenes Cas9 and Acidaminococcus sp. Cpf1(HEK293-Cas9-Cpf1 ), using the Nucleofector® 96-well Shuttle System(Lonza, Allendale, N.J.) and the following protocol. Cas9-Cpf1-NATNAsare prepared in an annealing buffer (1.25 mM HEPES, 0.625 mM MgCl₂,9.375 mM KCl at pH 7.5), incubated for 2 minutes at 95° C., removed froma thermocycler, allowed to equilibrate to room temperature, anddispensed in a 5 μL final volume in a 96-well plate. Culture medium isaspirated from HEK293-Cas9-Cpf1 cells, the cells are washed once withcalcium and magnesium-free PBS, and are then trypsinized by the additionof TrypLE (Life Technologies, Grand Island, N.Y.) followed by incubationat 37° C. for 3-5 minutes. Trypsinized cells are gently pipetted up anddown to form a single-cell suspension and added to DMEM complete culturemedium composed of DMEM culture medium (Life Technologies, Grand Island,N.Y.) containing 10% Fetal Bovine Serum (FBS; Thermo Fisher Scientific,Wilmington, Del.) and supplemented with penicillin and streptomycin(Life Technologies, Grand Island, N.Y.).

The cells are then pelleted by centrifugation for 3 minutes at 200×g,the culture medium aspirated, and cells re-suspended in PBS. The cellsare counted using the Countess® II Automated Cell Counter (LifeTechnologies, Grand Island, N.Y.). 2.2×10⁷ cells are transferred to a1.5 mL microfuge tube and pelleted. The PBS is aspirated and the cellsare re-suspended in Nucleofector™ SF (Lonza, Allendale, N.J.) solutionto a density of 1×10⁷ cells/mL. 20 μL of the cell suspension are thenadded to individual wells containing 5 uL of the Cas9-Cpf1-NATNA and theentire volume is transferred to the wells of a 96-well Nucleocuvette™Plate (Lonza, Allendale, N.J.). The plate is loaded onto theNucleofector™ 96-well Shuttle™ (Lonza, Allendale, N.J.) and cells arenucleofected using the 96-CM-130 Nucleofector™ program (Lonza,Allendale, N.J.). Post-nucleofection, 70 μL DMEM complete culture mediumare added to each well, and 50pL of the cell suspension are transferredto a collagen coated 96-well cell culture plate containing 150 μLpre-warmed DMEM complete culture medium. The plate is then transferredto a tissue culture incubator and maintained at 37° C. in 5% CO₂ for 48hours.

B. Double-Stranded DNA Target Sequence Generation for T7E1 Assay

gDNA is isolated from HEK293-Cas9-Cpf1 cells 48 hours after transfectionof the Cas9-Cpf1-NATNA using 504, QuickExtract DNA Extraction solution(Epicentre, Madison, Wis.) per well followed by incubation at 37° C. for10 minutes, 65° C. for 6 minutes and 95° C. for 3 minutes to stop thereaction. gDNA is then diluted with 150 μL water and samples are storedat −80° C.

DNA for T7E1 is generated by PCR amplification of double-stranded DNAtarget sequences (e.g., VEGFA and IDO2) from isolated gDNA. PCRreactions are set up using 80 μL gDNA as template with KAPA HiFi HotStart polymerase and contain 0.5 U of polymerase, lx reaction buffer,0.4mM dNTPs and 300nM forward and reverse primers directed to thedouble-stranded DNA target sequence (e.g., Example 2, Table 3; SEQ IDNO. 5 and SEQ ID NO. 6; and SEQ ID NO. 7 and SEQ ID NO. 8) in a totalvolume of 25 μL. The DNA target sequence is amplified using thefollowing conditions: 95° C. for 5 minutes, 4 cycles of 20 seconds at98° C., 20 seconds at 70° C., minus 2° C./cycle, 30 seconds at 72° C.,followed by 30 cycles of 15 seconds at 98° C., 20 seconds at 62° C., 20seconds at 72° C., and a final extension at 72° C. for 1 minute.

C. T7E1 Assay

PCR-amplified double-stranded DNA target sequences for T7E1 assays aredenatured at 95° C. for 10 minutes and then allowed to re-anneal bycooling to 25° C. at −0.5° C./s in a thermal cycler. The re-annealed DNAis incubated with 0.5 μL T7 Endonuclease I in 1× NEBuffer 2 buffer (NewEngland Biolabs, Ipswich, Mass.) in a total volume of 154, for 25minutes at 37° C. T7E1 reactions are analyzed using the FragmentAnalyzer™ System (Advanced Analytical Technologies, Ames, Iowa) and theDNF-910 Double-stranded DNA Reagent Kit (Advanced AnalyticalTechnologies, Ames, Iowa). The Fragment Analyzer™ System provides theconcentration of each cleavage fragment and of the double-stranded DNAtarget sequence that remains after cleavage.

Cleavage percentages of the double-stranded DNA target sequences arecalculated from the concentration of each cleavage fragment and thedouble stranded DNA target sequence that remains after cleavage hastaken place, using the following formula:

$\begin{matrix}{{\% \mspace{14mu} {cleavage}} = \left( {1 - \sqrt{\left( {1 - \frac{\left( {{{frag}\; 1} + {{frag}\; 2}} \right)}{\left( {{{frag}\; 1} + {{frag}\; 2} + {parent}} \right)}} \right)}} \right)} & {{EQUATION}\mspace{14mu} 1}\end{matrix}$

In Equation 1, frag1 and frag2 concentrations correspond to theconcentration of Cas9 cleavage fragments of the double-stranded DNAtarget sequence and parent corresponds to the double-stranded DNA targetsequence that remains after cleavage has taken place.

The T7E1 assay for detection of target sequence modifications ineukaryotic cells provides data to demonstrate that theCas9-Cpf1-NATNA/Cas9&Cpf1 protein complexes described herein facilitateCas9 and Cpf1-mediated site-specific in vivo cleavage of multipledouble-stranded DNA target sequences. sgRNA, crRNA and/or tracrRNA/crRNApolynucleotides having the same DNA target binding sequence as theCas9-Cpf1-NATNA can also be included in the assay to compare the Cas9-and Cpf1-mediated site-specific cleavage percentages between theconstructs.

Following the guidance of the present specification and Examples, theT7E1 assay described in this Example can be practiced by one of ordinaryskill in the art with other Type II CRISPR Cas9 proteins or Type VCRISPR Cpf1 proteins and their cognate Cas9-Cpf1-NATNA.

EXAMPLE 8 Probing for Sites Tolerant of Modification in Class 2 Type VGuide crRNA Backbones

This Example describes the generation and testing of variousmodifications of Class 2 Type V guide crRNAs and their suitability foruse in constructing cross-type-NATNAs.

In this Example, crRNA backbone sequences were modified, and themodified crRNA were tested with a cognate Cpf1 nuclease to facilitateidentification of regions or positions in the Cpf1-crRNA backbonewherein a Cas9-sgRNA can be linked through hydrogen base-pair bonds or acovalent bond (e.g., phosphodiester bond).

Various regions in the F. novicida Cpf1-crRNA were selected formodification. Primers for the generation of modified Cpf1-crRNA wereordered from a commercial manufacturer. Double-stranded DNA templatesfor crRNA transcription were generated as previously describe (e.g.,Example 1). Sequences used for crRNA template assembly are shown inTable 7.

TABLE 7 crRNA Template Assembly Primers Target sequence crRNA sequencePrimers used for spacer modification PCR assembly VEGFA wild type crRNASEQ ID Nos. 1, 17, 27 IDO2 wild type crRNA SEQ ID Nos. 1, 18, 4 VEGFAsingle-stranded 5′ tail SEQ ID Nos. 1, 19, 27 IDO2 single-stranded 5′tail SEQ ID Nos. 1, 20, 4 VEGFA increased pseudo-knot length SEQ ID Nos.1, 21, 27 IDO2 increased pseudo-knot length SEQ ID Nos. 1, 22, 4 VEGFA3′ hairpin addition SEQ ID Nos. 1, 23, 27 IDO2 3′ hairpin addition SEQID Nos. 1, 24, 4 VEGFA 5′ hairpin addition SEQ ID Nos. 1, 25, 27 IDO2 5′hairpin addition SEQ ID Nos. 1, 26, 4

FIG. 14A illustrates the wild-type Cpf1-crRNA. FIG. 14B illustrates thewild-type Cpf1 pre-crRNA. Examples of modifications made to Cpf1-crRNAare diagrammed in FIG. 14C, FIG. 14D, and FIG. 14E, wherein themodifications are indicated by the non-shaded portions of the diagrammedmodified Cpf1-crRNA. The corresponding sequences for the Cpf1-crRNAs arepresented in Table 8, including the spacer, type of modification,(modified) crRNA sequence, and SEQ ID. NO. for the (modified) crRNAsequence, as well as the figure corresponding to general structure foreach (modified) crRNA.

TABLE 8 Modified Cpf1-crRNA Sequences crRNA Target sequence sequencemodifi- Modified crRNA SEQ ID spacer cation sequence NO. FIG. VEGFAwild type GAAUUUCUACUGUUGUAGA SEQ ID 14A crRNA UGAAAGGGGGUGGGGGGAGNO. 28 UUUGCU IDO2 wild type GAAUUUCUACUGUUGUAGA SEQ ID 14A crRNAUGGUAGAGAAAGAAGCAGU NO. 29 GCCUGG VEGFA single- GGUCUAAGAACUUUAAAUASEQ ID 14B stranded AUUUCUACUGUUGUAGAUG NO. 30 5′ tailAAAGGGGGUGGGGGGAGUU UGCU IDO2 single- GGUCUAAGAACUUUAAAUA SEQ ID 14Bstranded AUUUCUACUGUUGUAGAUG NO. 31 5′ tail GUAGAGAAAGAAGCAGUGC CUGGVEGFA increased GAAUUUCUACUGUUCACUG SEQ ID 14C pseudo-knotCCGUAUAGGCAGUGAACUU NO. 32 length GUAGAUGAAAGGGGGUGGG GGGAGUUUGCU IDO2increased GAAUUUCUACUGUUCACUG SEQ ID 14C pseudo-knot CCGUAUAGGCAGUGAACUUNO. 33 length GUAGAUGGUAGAGAAAGAA GCAGUGCCUGG VEGFA 3′ hairpinGAAUUUCUACUGUUGUAGA SEQ ID 14D addition UGAAAGGGGGUGGGGGGAG NO. 34UUUGCUGCCAGGUUCACUG CCGUAUAGGCAG IDO2 3′ hairpin GAAUUUCUACUGUUGUAGASEQ ID 14D addition UGGUAGAGAAAGAAGCAGU NO. 35 GCCUGGGCCAGGUUCACUGCCGUAUAGGCAG VEGFA 5′ hairpin GGUUCACUGCCGUAUAGGC SEQ ID 14E additionAGGCAAGAAUUUCUACUGU NO. 36 UGUAGAUGAAAGGGGGUGG GGGGAGUUUGCU IDO2 5′hairpin GGUUCACUGCCGUAUAGGC SEQ ID 14E addition AGGCAAGAAUUUCUACUGUNO. 37 UGUAGAUGGUAGAGAAAGA AGCAGUGCCUGG

S. pyogenes Cas9 protein and F. novicida Cpf1 protein were recombinantlyexpressed in E. coli and purified for use in in vitro biochemicalcleavage assays. VEGFA and IDO2 double-stranded DNA target sequenceswere generated as described in Example 2. Biochemical cleavage ofPCR-generated target sequences were carried out essentially as describedin Example 3, with the modification that only the F. novicida Cpf1 wasused.

FIG. 15 presents the results of the F. novicida Cpf1 biochemicalcleavage assay using the modified Cpf1-crRNAs (Table 8). The y-axisshows percent cleavage of a double-stranded DNA target sequence, and thex-axis shows the SEQ ID NO. corresponding to the (modified) Cpf1-crRNAused in the assay. As can be seen from the data, inclusion of additionalRNA bases and secondary structures (e.g., hairpin elements) wastolerated in the Cpf1-crRNA.

The methods described in this Example can be used to identify locationsin a Cpf1-crRNA sequence that permit engineering of the Cpf1 crRNA bylinkage to a Cas9 crRNA, tracrRNA, or sgRNA to generate aCas9-Cpf1-NATNA. Cpf1 crRNA from different species may exhibit differentdegrees of tolerance for various sequence modification. Additionally,different Cas9 crRNA, tracrRNA, or sgRNA may be more amenable forinclusion into a Cas9-Cpf1-NATNA.

Cpf1 crRNAs that are capable of mediating cleavage of a DNA targetsequence with their cognate Cpf1 proteins can be validated for activityin cells using the method described in Example 4.

This Example can be practiced by one of ordinary skill in the art withother Class 2 Type V CRISPR proteins and their cognate Type V CRISPRguide crRNA. Following the guidance of the present specification andExamples, the modification of a Cpf1-crRNA (e.g., introduction ofvarious sequence and secondary structural modifications, and/or deletionof various sequences) can be used to probe for locations for insertionor linkages to facilitate making a Cas9-Cpf1-NATNA.

EXAMPLE 9 Probing for Sites Tolerant of Modification in Class 2 Type IICas9 Guide RNA Backbones

This Example describes the generation and testing of variousmodifications of Class 2 Type II guide RNA(s) and their suitability foruse in constructing cross-type-NATNAs.

In this Example, modifications are introduced into the RNA backbone ofClass 2 Type II CRISPR guide RNAs to identify locations for engineeringor attachment of various nucleic acid sequences. The method describedbelow is adapted from Briner, A., et al., Molecular Cell 56(2):333-339(2014). Not all of the following steps are required for screening normust the order of the steps be as presented.

A sgRNA from a Class 2 Type II CRISPR system is selected forengineering. The guide RNA sequence is modified in silico to introducebase substitution(s) in region(s) selected from one or more of thefollowing: the lower stem, bulge, upper stem, tetraloop, nexus, linkingsequences, and 3′ hairpins. Base substitution can be used to introducemismatches in the hydrogen base-pair interactions of any of the guideRNA regions, or base-pair mutation may preserve hydrogen base-pairinteraction through substitution of two bases introducing alternativeWatson-Crick base pairs. Substitution of bases can also be used tointroduce novel hydrogen base-pair interaction within the guide RNAbackbone (e.g., within the bulge sequence).

Regions of the guide RNA can be independently engineered to introducesecondary structure elements into the guide RNA backbone. Suchmodifications include hairpins, pseudo-knots, ribozymes, or othermodification known to one skilled in the art. Furthermore, the guide RNAbackbone can be modified to delete portions of the guide RNA backbone,either through deletion at the 5′ end, 3′ end or internal to the guideRNA.

In silico designed Class 2 Type II CRISPR guide RNA sequences areprovided to a commercial manufacturer for synthesis.

Modified Class 2 Type II CRISPR guide RNAs are evaluated for theirability to support cleavage of a double-stranded DNA target sequencemediated by their cognate Cas9 proteins. Double-stranded DNA targetsequences and biochemical cleavage assay are carried out in a mannersimilar to those described in Example 2 and Example 3. Guide RNAs thatare capable of mediating cleavage of a DNA target sequence with theircognate Cas9 proteins can be validated for activity in cells using themethod described in Example 4.

Following the guidance of the present specification and Examples, themodification of a Cas9 guide RNA(s) (e.g., introduction of varioussequence and secondary structural modifications, and/or deletion ofvarious sequences) can be used to probe for locations for insertion orlinkages to facilitate making a Cas9-Cpf1-NATNA. This Example can bepracticed by one of ordinary skill in the art with other Type II CRISPRCas9 proteins and other Type II CRISPR guide RNA.

EXAMPLE 10

Screening of Cross-type-NATNAs Comprising DNA Target Binding Sequences

This Example illustrates the use of cross-type-NATNAs of the presentinvention to modify DNA target sequences present in human genomic DNAand to measure the level of cleavage activity at those sites.

Target sites (DNA target sequences) are first selected from genomic DNA.Cas9-Cpf1-NATNAs are designed to target the selected sequences. Assays(e.g., as described in Example 3) are performed to determine the levelof DNA target sequence cleavage.

Not all of the following steps are required for every screening nor mustthe order of the steps be as presented, and the screening can be coupledto other experiments, or form part of a larger experiment.

A. Selecting DNA Target Regions (DNA Target Sequences) from Genomic DNA

All PAM sequences (e.g., NGG, TTTN) are identified within the selectedgenomic region.

One or more 20 nucleotide sequences (Cas9 DNA target sequence) that are5′ adjacent to a NGG PAM sequence are identified and selected.

One or more 24 nucleotide sequences (Cpf1 DNA target sequence) that are3′ adjacent to a TTTN PAM sequence are identified and selected.

Criteria for selection of nucleic acid target sequences can include, butare not limited to, the following: homology to other regions in thegenome; percent G-C content; melting temperature; presences ofhomopolymer within the spacer; distance between the two sequences; andother criteria known to one skilled in the art.

The Cas9 DNA target binding sequence is appended to the 5′ end of anappropriate Cas9-Cpf1-NATNA, and the Cpf1 DNA target binding sequence isappended to the 3′ end of the same Cas9-Cpf1-NATNA (e.g., aCas9-Cpf1-NATNA as illustrated in FIG. 4). A Cas9-Cpf1-NATNA constructis typically synthesized by a commercial manufacturer, and is based onsequences provided to the manufacturer. Alternatively, theCas9-Cpf1-NATNA construct is produced as described in Example 1 by invitro transcription.

A Cas9-Cpf1-NATNA, as described herein, is used with cognate Class 2Type II and Class 2 Type V CRISPR Cas proteins (e.g., theCas9-associated nucleic acids of a Cas9-Cpf1-NATNA bind their cognateCas9 protein, and the Cpf1-associated nucleic acids of a Cas9-Cpf1-NATNAbind their cognate Cpf1 protein).

B. Determination of Cleavage Percentages and Specificity

In vitro cleavage percentages and specificity (e.g., the amount ofoff-target binding) related to a Cas9-Cpf1-NATNA are determined, forexample, using the cleavage assays described in Example 3, and arecompared as follows:

(1) If only a single pair of DNA target sequences are identified orselected for a Cas9-Cpf1-NATNA, the cleavage percentage and specificityfor each of the DNA target sequences are determined. If so desired,cleavage percentage and/or specificity are altered in furtherexperiments using methods including, but not limited to, modifying theCas9-Cpf1-NATNA, or introducing effector proteins/effectorprotein-binding sequences to modify the Cas9-Cpf1-NATNA or one or bothof the Cas proteins, or ligand/ligand binding moieties to modify theCas9-Cpf1-NATNA or one or both of the Cas proteins.

(2) If multiple pairs of DNA target sequences are identified or selectedfor a Cas9-Cpf1-NATNA, the percentage cleavage data and site-specificitydata obtained from the cleavage assays are compared between differentDNAs comprising the target binding sequence to identify the DNA targetsequences having the desired cleavage percentage and specificity.Cleavage percentage data and specificity data provide criteria on whichto base choices for a variety of applications. For example, in somesituations the activity of the Cas9-Cpf1-NATNAs may be the mostimportant factor. In other situations, the specificity of the cleavagesite may be relatively more important than the cleavage percentage. Ifso desired, cleavage percentage and/or specificity are altered infurther experiments using methods of the present invention including,but not limited to, modifying the Cas9-Cpf1-NATNA, or introducingeffector proteins/effector protein-binding sequences to modify theCas9-Cpf1-NATNA or one or both of the Cas proteins, or ligand/ligandbinding moieties to modify the Cas9-Cpf1-NATNA or one or both of the Casproteins.

Alternatively, or in addition to the in vitro analysis, in cell cleavagepercentages and specificities associated with Cas9-Cpf1-NATNAs obtainedusing, for example, the method described in Example 4, are compared asfollows:

(1) If only a single pair of DNA target sequences are identified orselected for a Cas9-Cpf1-NATNA, the cleavage percentage and specificityfor each of the DNA target sequences are determined. If so desired,cleavage percentage and/or specificity are altered in furtherexperiments using including, but not limited to, modifying theCas9-Cpf1-NATNA, or introducing effector proteins/effectorprotein-binding sequences to modify the Cas9-Cpf1-NATNA or one or bothof the Cas proteins, or ligand/ligand binding moieties to modify theCas9-Cpf1-NATNA or one or both of the Cas proteins.

(2) If multiple pairs of DNA target sequences are identified or selectedfor a Cas9-Cpf1-NATNA, the percentage cleavage data and site-specificitydata obtained from the cleavage assays are compared between differentDNAs comprising the target binding sequences to identify the DNA targetsequences having the desired cleavage percentage and specificity.Cleavage percentage data and specificity data provide criteria on whichto base choices for a variety of applications. For example, in somesituations the activity of the Cas9-Cpf1-NATNAs may be the mostimportant factor. In other situations, the specificity of the cleavagesite may be relatively more important than the cleavage percentage. Ifso desired, cleavage percentage and/or specificity are altered infurther experiments using methods of the present invention including butnot limited to modifying the Cas9-Cpf1-NATNA, or introducing effectorproteins/effector protein-binding sequences to modify theCas9-Cpf1-NATNA or one or both of the Cas proteins, or ligand/ligandbinding moieties to modify the Cas9-Cpf1-NATNA or one or both of the Casproteins.

Following the guidance of the present specification and Examples, thescreening described in this Example can be practiced by one of ordinaryskill in the art with other cross-type-NATNAs for use with cognate Class2 Type II CRISPR Cas9 proteins and Class 2 Type V CRISPR Cpf1 proteins.

As is apparent to one of skill in the art, various modification andvariations of the above embodiments can be made without departing fromthe spirit and scope of this invention. Such modifications andvariations are within the scope of this invention.

1-20. (canceled)
 21. A method of binding a first nucleic acid targetsequence, a second nucleic acid target sequence, and a third nucleicacid target sequence, comprising: contacting the first nucleic acidtarget sequence, the second nucleic acid target sequence, and the thirdnucleic acid target sequence with a nucleic acid/protein compositioncomprising a CRISPR Type II Cas9 protein; a first CRISPR Type V Cpf1protein; a second CRISPR Type V Cpf1 protein; and an engineered CRISPRClass 2 cross-type-nucleic-acid targeting nucleic acid (“CRISPR Class 2cross-type NATNA”), comprising a first CRISPR Type V Cpf1-associatednucleic-acid targeting nucleic acid, having a 5’ end and a 3′ end,comprising a first spacer element complementary to the first nucleicacid target sequence (“first Cpf1-NATNA”); a second CRISPR Type VCpf1-associated nucleic-acid targeting nucleic acid, having a 5′ end anda 3′ end, comprising a second spacer element complementary to the secondnucleic acid target sequence (“second Cpf1-NATNA”); a first CRISPR TypeII Cas9-associated nucleic-acid targeting nucleic acid, having a 5′ endand a 3′ end, comprising a third spacer element complementary to thethird nucleic acid target sequence (“first Cas9-NATNA”); and a secondCRISPR Type II Cas9-associated nucleic-acid targeting nucleic acid,having a 5′ end and a 3′ end, comprising a tracr element (“secondCas9-NATNA”); wherein the 3′ end of the first Cas9-NATNA is covalentlyconnected through a loop element with the 5’ end of the secondCas9-NATNA, resulting in a single-Cas9-associated nucleic-acid targetingnucleic acid (“single-Cas9-NATNA”), having a 5′ end and a 3′ end; andwherein the single-Cas9-NATNA is connected with the first Cpf1-NATNA andthe second Cpf1-NATNA; thereby facilitating binding of the nucleicacid/protein composition to the first nucleic acid target sequence, thesecond nucleic acid target sequence, and the third nucleic acid targetsequence.
 22. The method of claim 21, wherein the method is carried outin vitro, in a cultured cell, or in a non-human subject.
 23. The methodof claim 21, wherein at least one of the Cpf1-NATNA, the firstCas9-NATNA, or the second Cas9-NATNA comprises RNA.
 24. The method ofclaim 23, wherein at least one of the Cpf1-NATNA, the first Cas9-NATNA,or the second Cas9-NATNA comprises DNA.
 25. The method of claim 21,wherein the first CRISPR Type V Cpf1 protein and the second CRISPR TypeV Cpf1 protein are both catalytically active and the CRISPR Type II Cas9protein is catalytically inactive, and the binding of the nucleicacid/protein composition to the first nucleic acid target sequence, thesecond nucleic acid target sequence, and the third nucleic acid targetsequence results in cutting of the first nucleic acid target sequenceand the second nucleic acid target sequence by the first and secondcatalytically active CRISPR Type V Cpf1 proteins, respectively; or thefirst CRISPR Type V Cpf1 protein and the second CRISPR Type V Cpf1protein are both catalytically inactive and the CRISPR Type II Cas9protein is catalytically active, and the binding of the nucleicacid/protein composition to the first nucleic acid target sequence, thesecond nucleic acid target sequence, and the third nucleic acid targetsequence results in cutting of the third nucleic acid target sequence bythe catalytically active CRISPR Type II Cas9 protein; or the firstCRISPR Type V Cpf1 protein is catalytically active, the second CRISPRType V Cpf1 protein is catalytically inactive, and the CRISPR Type IICas9 protein is catalytically inactive, and the binding of the nucleicacid/protein composition to the first nucleic acid target sequence, thesecond nucleic acid target sequence, and the third nucleic acid targetsequence results in cutting of the first nucleic acid target sequence bythe first catalytically active CRISPR Type V Cpf1 protein; or the firstCRISPR Type V Cpf1 protein and the second CRISPR Type V Cpf1 protein areeach catalytically active, and the CRISPR Type II Cas9 protein iscatalytically active, and the binding of the nucleic acid/proteincomposition to the first nucleic acid target sequence, the secondnucleic acid target sequence, and the third nucleic acid target sequenceresults in cutting of the first nucleic acid target sequence, the secondnucleic acid target sequence, and the third nucleic acid target sequenceby the first and second catalytically active CRISPR Type V Cpf1 proteinsand the catalytically active CRISPR Type II Cas9 protein, respectively.26. The method of claim 21, wherein the nucleic acid/protein compositionfurther comprises at least one donor polynucleotide, the donorpolynucleotide comprising: a nucleotide sequence complementary to thefirst or second spacer element of the first or second Cpf1-NATNA,respectively, if the first and second CRISPR Type V Cpf1 proteins areeach catalytically inactive; or a nucleotide sequence complementary tothe third spacer element of the single-Cas9-NATNA if the CRISPR Type IICas9 protein is catalytically inactive; and the at least one donorpolynucleotide is capable of associating with the first, second, orthird spacer element through hydrogen bonding.
 27. The method of claim26, wherein the donor polynucleotide comprises single-stranded DNA. 28.The method of claim 21, wherein the first nucleic acid target sequence,the second nucleic acid target sequence, and the third nucleic acidtarget sequence are a single polynucleotide.
 29. The method of claim 28,wherein the single polynucleotide comprises genomic DNA.
 30. The methodof claim 21, wherein the first nucleic acid target sequence, the secondnucleic acid target sequence, and the third nucleic acid target sequenceare a first polynucleotide, a second polynucleotide, and a thirdpolynucleotide, respectively.
 31. A method of binding a first nucleicacid target sequence and a second nucleic acid target sequence,comprising: contacting the first nucleic acid target sequence and thesecond nucleic acid target sequence with a nucleic acid/proteincomposition comprising a CRISPR Type II Cas9 protein; a CRISPR Type VCpf1 protein; and an engineered CRISPR Class 2 cross-type-nucleic-acidtargeting nucleic acid (“CRISPR Class 2 cross-type-NATNA”), comprising afirst CRISPR Type II Cas9-associated nucleic-acid targeting nucleicacid, having a 5’ end and a 3′ end, comprising a first spacer elementcomplementary to the first nucleic acid target sequence (“firstCas9-NATNA”); a second CRISPR Type II Cas9-associated nucleic-acidtargeting nucleic acid, having a 5′ end and a 3′ end, comprising a tracrelement (“second Cas9-NATNA”) and a first linker element, having a 5′end and a 3′ end, wherein the 3′ end of the first linker element iscovalently connected at the 5′ end of the second Cas9-NATNA; a CRISPRType V Cpf1-associated nucleic-acid targeting nucleic acid, having a 5′end and a 3′ end, comprising a second spacer element complementary tothe second nucleic acid target sequence (“Cpf1-NATNA”), wherein the 3’end is covalently connected with the 5′ end of the first linker element;and a second linker element, having a 5′ and a 3′ end, wherein thesecond linker element is covalently connected at the 5′ end of the firstCas9-NATNA or at the 3′ end of the first Cas9-NATNA or at the 3′ end ofthe second Cas9-NATNA; thereby facilitating binding of the nucleicacid/protein composition to the first nucleic acid target sequence andthe second nucleic acid target sequence.
 32. The method of claim 31,wherein the method is carried out in vitro, in a cultured cell, or in anon-human subject.
 33. The method of claim 31, wherein at least one ofthe Cpf1-NATNA, the first Cas9-NATNA, or the second Cas9-NATNA comprisesRNA.
 34. The method of claim 33, wherein at least one of the Cpf1-NATNA,the first Cas9-NATNA, or the second Cas9-NATNA comprises DNA.
 35. Themethod of claim 31, wherein the CRISPR Type V Cpf1 protein iscatalytically inactive and the CRISPR Type II Cas9 protein iscatalytically active, and the binding of the nucleic acid/proteincomposition to the first nucleic acid target sequence and the secondnucleic acid target sequence results in cutting of the first nucleicacid target sequence by the catalytically active CRISPR Type II Cas9protein; or the CRISPR Type V Cpf1 protein is catalytically active andthe CRISPR Type II Cas9 protein is catalytically inactive, and thebinding of the nucleic acid/protein composition to the first nucleicacid target sequence and the second nucleic acid target sequence resultsin cutting of the second nucleic acid target sequence by thecatalytically active CRISPR Type V Cpf1 protein; or the CRISPR Type VCpf1 protein is catalytically active and the CRISPR Type II Cas9 proteinis catalytically active, and the binding of the nucleic acid/proteincomposition to the first nucleic acid target sequence and the secondnucleic acid target sequence results in cutting of the first nucleicacid target sequence and the second nucleic acid target sequence by thecatalytically active CRISPR Type V Cpf1 protein and the catalyticallyactive CRISPR Type II Cas9 protein, respectively.
 36. The method ofclaim 31, wherein the nucleic acid/protein composition further comprisesa donor polynucleotide, the donor polynucleotide comprising: anucleotide sequence complementary to the second spacer element of theCpf1-NATNA if the CRISPR Type V Cpf1 protein is catalytically inactive,or a nucleotide sequence complementary to the first spacer element ofthe first Cas9-NATNA if the CRISPR Type II Cas9 protein is catalyticallyinactive; and the donor polynucleotide is capable of associating withthe first or second spacer element through hydrogen bonding.
 37. Themethod of claim 36, wherein the donor polynucleotide comprisessingle-stranded DNA.
 38. The method of claim 31, wherein the firstnucleic acid target sequence and the second nucleic acid target sequenceare a single polynucleotide.
 39. The method of claim 38, wherein thesingle polynucleotide comprises genomic DNA.
 40. The method of claim 31,wherein the first nucleic acid target sequence and the second nucleicacid target sequence are a first polynucleotide and a secondpolynucleotide, respectively.